Nucleotide and polypeptide sequences of pestivirus strains

ABSTRACT

The invention provides a nucleotide sequence corresponding to a classical swine fever virus (CSFV) genome or a part or a mutant thereof, which comprises at least a part of the nucleotide sequence of the CSFV C-strain depicted in SEQ ID No. 1, or a complement or RNA equivalent of such nucleotide sequence, or which comprises a nucleotide sequence encoding at least the amino acid sequence 268-494 of the amino acid sequence depicted in SEQ ID No. 1, or a complement or RNA equivalent of such nucleotide sequence. Also provided is a pestivirus polypeptide corresponding to the amino acid sequence 690-1063 of SEQ ID No. 1 or part thereof, which contains a mutation in one of the epitopes within amino acid sequences 691-750 or 785-870, said mutation altering said epitope. Further provided is a method of determining the presence of a test substance capable of specifically binding with a binding site of a binding partner, in a sample, by means of competition of said test substance with a measurable amount of a reference substance capable of specifically binding with the same binding site of said binding partner, comprising: (1) contacting said sample with (a) said reference substance bound to a solid carrier, (b) the binding partner of said reference substance, said binding partner molecule containing at least two identical binding sites for said reference substance, and (c) said reference substance provided with a label; (2) measuring the degree of binding of said label to said carrier.

This application is a continued prosecution application of an application of the same serial number filed Aug. 17, 1998, now abandoned, which was a continued prosecution application of an application of the same serial number, which was the U.S. national phase of international application PCT/NL95/00214, filed Jun. 16, 1995, the national stage application being now abandoned and having a §102(e) date of Dec. 17, 1996.

FIELD OF THE INVENTION

The invention discloses a method for the construction of a full-length DNA copy of the genome of the C-strain, a classical swine fever vaccine strain, and transcription of RNA thereof which after transfection in cells gives rise to synthesis of infectious C-strain virus. The invention also comprises C-strain derived (pestivirus) vaccines, as well as subunit vaccines against pestivirus and diagnostic means and methods in relation to pestivirus infections. The invention furthermore provides a method of detecting an immunoactive substance in a sample by means of a competitive assay.

BACKGROUND OF THE INVENTION

Classical swine fever (CSF) or hog cholera is a highly contagious and often fatal disease of pigs which is characterised by fever and haemorrhages and can run an acute or chronic course (Van Oirschot. 1986. Hog cholera, p. 289-300. In Diseases of Swine. Iowa State University Press, Ames). Outbreaks of the disease occur intermittently in several European and other countries and can cause large economic losses.

Vaccination of pigs with a live attenuated Classical swine fever virus (CSFV) vaccine strain, the “Chinese” strain (C-strain), protects pigs against CSF (Terpstra and Wensvoort. 1988. Vet. Microbiol. 16: 123-128). A major drawback of vaccinating pigs with the conventional vaccines, of which the C-strain is one, is that these vaccinated pigs cannot be distinguished serologically from pigs infected with a CSFV field strain. The C-strain, however, is considered one of the most effective and safe live vaccines. Addition of a (serological) marker to the C-strain would be highly advantageous and would improve the vaccine.

CSFV is a member of the Pestivirus genus of the Flaviviridae (Francki, R. I. B. et al. 1991. Flaviviridae, p. 223-233. In Fifth report of the International Committee on Taxonomy of Viruses. Archiv. Virol. Suppl. 2, Springer Verlag, Vienna). The other two members of the Pestivirus genus, which are structurally, antigenically and genetically closely related to CSFV, are Bovine viral diarrhoea virus (BVDV) mainly affecting cattle, and Border disease virus (BDV) mainly affecting sheep (Moennig and Plagemann, 1992. Adv. Virus Res. 41: 53-98; Moormann et al., 1990. Virology 177: 184-198; Becher et al. 1994. Virology 198: 542-551).

The genomes of pestiviruses consist of a positive strand RNA molecule of about 12.5 kb (Renard et al. 1985. DNA 4: 429-438; Moormann and Hulst 1988. Virus Res. 11: 281-291; Becher et al. 1994. Virology 198: 542-551). The positive strand RNA genomes of several non-cytopathogenic BVDV strains, however, may be considerably larger (Meyers et al. 1991. Virology 180: 602-616; Meyers et al. 1992. Virology 191: 368-386; Qi et al. 1992. Virology 189: 285-292).

An inherent property of viruses with a positive strand RNA genome is that their genomic RNA is infectious, i.e. after transfection of this RNA in cells that support viral replication infectious virus is produced. As expected, the genomic (viral) RNA of pestiviruses is also infectious (Moennig and Plagemann, 1992. Adv. Virus Res. 41: 53-98).

For several years recombinant DNA technology has allowed in vitro transcription of cloned DNA. This possibility has opened the way to synthesize infectious RNA in vitro from a DNA copy of the genome of a positive strand RNA virus. It is well known in the field of molecular engineering that DNA, in contrast to RNA, is easily manipulated by site directed mutagenesis. Hence, the availability of the technique to produce synthetic infectious RNA has greatly enhanced the study of e.g. replication, virulence, pathogenesis, RNA recombination, vector development, and antiviral strategies of the positive strand RNA viruses. However, application of the technology may cause severe problems. The nature of these problems has been described in a recent review by Boyer and Haenni. 1994. (Virology 198: 415-426). In fact, the success or failure to construct a full-length DNA copy of the genome of a positive strand RNA virus and to produce synthetic infectious RNA from such a full-length DNA copy cannot be reliably predicted.

SUMMARY OF THE INVENTION

The invention provides nucleotide sequences corresponding to a CSFV genome which comprise at least a part of the nucleotide sequence of the CSFV C-strain depicted in SEQ ID No. 1, or a complement or RNA equivalent of such nucleotide sequence, or mutants thereof. Also provided are degenerate nucleotide sequences having different nucleotides but encoding the same amino acids. The invention also covers potypeptides encoded by these nucleotide sequences, and vaccine strains, the genome of which contains such a nucleotide sequence, in particular a recombinant virus strain based on transcripts of a full-length DNA copy of the genome of the CSFV C-strain.

Partial nucleotide sequences as indicated above are also useful, in particular those which contain a mutation in the structural region of the virus genome, i.e. in the nucleotide sequence encoding amino acids 1-1063 of the sequence depicted in SEQ ID No. 1. The mutation may be a substitution by a corresponding part of the genome of another pestivirus strain, a substitution of one or amino acids, or a deletion. The mutation may also be an inserted or substituted heterologous nucleotide sequence altering the translation strategy of the CSFV nucleotide sequence or altering the processing of a polypeptide encoded by the CSFV nucleotide sequence. Furthermore, the mutation may be an inserted or substituted heterologous nucleotide sequence encoding a polypeptide inducing immunity against another pathogen; in this case the CSFV sequence is used as a vector for heterologous immunogens.

The invention is also concerned with nucleotide sequences of a pestivirus genome in general or a part or a mutant thereof, which sequences contain a mutation in a subregion of the E1 protein, i.e. in the nucleotide sequence encoding amino acids corresponding to amino acids 691-750 or 785-870 of the sequence depicted in SEQ ID No. 1, as well as the polypeptides encoded by these nucleotide sequences. These polypeptides are particularly useful for protecting animals against a pestivirus infection in such a way so as to allow a diagnosis which distinguishes between animals infected with field strains of the pestivirus and vaccinated animals.

The invention is furthermore concerned with vaccines containing a nucleotide sequence, a polypeptide, or a vaccine strain as indicated above, as well as to diagnostic compositions containing a nucleotide sequence or a polypeptide as mentioned above, or an antibody raised against such polypeptide.

The invention also relates to methods and means for diagnosis of pestivirus infections, especially with such means and methods which distinguish between infected animals infected and vaccinated animals.

The invention also provides a method for determining test substances, such as an antibody or an antigen in an immunoassay, by means of a specific binding test, wherein a specifically binding reference substance in immobilised form and the same specifically binding reference substance in labeled form are used.

DETAILED DESCRIPTION OF THE INVENTON

The invention provides the complete cDNA sequence of the RNA genome of the “Chinese” strain (C-strain; EP-A-351901) of CSFV. This allows the construction of a full-length DNA copy of this sequence, of which synthetic RNA can be transcribed that after transfection in suitable cells, such as SK6-M cells (Kasza, L. et al. 1972. Res. Vet. Sci., 13: 46-51; EP-A-351901) gives rise to synthesis of infectious C-strain virus. The use of this finding for the development of modified C-strain vaccines, e.g. vaccines which contain a (serological) marker, is described. Although the invention is illustrated for one CSFV strain, it is also applicable and useful for other pestivirus strains by exchanging specific genomic segments, described below, between the other pestivirus and the CSFV C-strain, or by constructing an “infectious”, DNA copy of the other pestivirus.

The nucleotide sequence of a DNA copy of the genomic RNA of the C-strain is depicted in SEQ ID No. 1. The numerals mentioned in the text are all related to this sequence and may differ slightly in the sequences of other pestiviruses. The nucleotide sequence is 12,311 nucleotides in length and contains one large open reading frame (ORF) of 11,694 nucleotides encoding a polyprotein of 3,898 amino acids (SEQ ID NO. 2). The size of this ORF is the same as that of the genomes of CSFV strains Brescia (Moormann et al. 1990. Virology 177: 184-198) and Alfort (Meyers et al. 1989. Virology 171: 555-567).

The ORF starts with the ATG at nucleotide positions 374 to 376 and stops at the TGA codon at nucleotide positions 12,068 to 12,070. The 5′ non-coding region which precedes the ORF is 373 nucleotides in length. Its sequence is highly conserved between strains Brescia, Alfort and C (SEQ ID NOS:3-5 in FIG. 2A), and the predicted secondary structure of this region resembles that of the 5′non-coding region of hepatitis C virus (Brown et al. 1992. Nucleic Acids Res. 20: 5041-5045), another member of the Flaviviridae. The 5′ non-coding region of hepatitis C virus has been shown to contain an internal ribosome entry site (Tsukiyama-Kohara et al. 1992. J. Virol. 66:1476-1483). Such sites have important regulatory functions (Agol. 1991. Adv. Virus. Res. 40:103-180). The analogy with hepatitis C virus indicates that the 5′non-coding region of CSFV also contains an internal ribosome entry site, which is located approximately between nucleotides 124 and 374 of the sequence of SEQ ID No. 1, as important regulatory element. The internal ribosome entry site may be used as a site for mutation in order to attenuate the virus, as well as for altering the translation strategy of the ORF.

A second important region regulating replication of pestiviruses is the 3′ non-coding region. Upon alignment of the C-strain sequence with the sequences of strains Brescia and Alfort, a sequence of 13 nucleotides unique to the C-strain was observed in this region (SEQ ID NOS:6-8 in FIG. 2B). This unique sequence TTTTCTTTTTTTT (SEQ ID NO:9) is located from nucleotide positions 12,128 to 12,140 in the sequence of SEQ ID No. 1. It is the only insertion of more than two nucleotides in a row observed in the sequence of the C strain compared to the sequences of strains Brescia and Alfort. For the rest, the sequences in the 3′ non-cooding regions of three CSFV strains are highly homologous. The overall homology between sequences in this region is lower when CSFV strains and BVDV strains are compared. Nevertheless, it is clear that the TTTTCTTTTTTTT sequence of the C-strain is also absent in the sequences of the 3′ non-coding regions of the BVDV strains. The TTTTCTTTTTTTT sequence therefore appears to be unique to the genome of the C-strain, and will provide an excellent marker for a C-strain specific sequence. This sequence can be used as a basis for nucleotide probes, and for sequence determination, to identify C-strain specific pestiviruses. Therefore, all pestivirus strains having this sequence in their 3′ non-coding region (not necessarily at an identical position as in the C-strain) are considered related to the C-strain, and are also part of the invention.

A crucial parameter for infectivity of transcripts of a DNA copy of the genome of a pestivirus is the amino acid sequence. In this respect, two aspects regarding the cloning and sequencing of RNA viruses in general, and pestiviruses in particular, had to be considered. First, the mutation frequency of the genome of positive strand RNA viruses is high (about 1/10⁴ nucleotides during replication), and therefore no stock of virus or viral RNA preparation is ever clonal with regard to the viral RNA it contains. Among these RNA molecules there may also be molecules which are noninfectious. If this were caused by premature stop codons in the large open reading frame, this would be easily recognised. Also mutations affecting active sites of viral enzymes, or known structures of proteins would be recognizable. However, where the relation between the amino acid sequence and the function and structure of a protein is unknown, which is the case with most of the pestivirus proteins, it is impossible to predict which amino acid is valid and which one is not. Second, mutations may have been introduced during cDNA synthesis. Therefore, the genome of the C-strain was cloned and sequenced independently twice. Regions with discrepancies between the sequences were cloned and sequenced at least thrice (compare FIG. 1). The sequence which was encountered twice at a particular position was regarded as the correct one at that position. The necessity of this approach for the generation of infectious transcripts of a DNA copy of the genome of the C-strain is demonstrated by the following finding. Full-length DNA copy pPRKflc-113, composed after the second round of cloning and sequencing (FIG. 3), appeared to be noninfectious. After cloning and sequencing of regions with discrepancies between the sequences of cDNA clones of the first and second round, there appeared to be five amino acids which were different in the full-length copy of the second round cDNA clones and the sequence of the C-strain considered correct. After correction of these five amino acids in pPRKflc-113, clone pPRKflc-133 was obtained which generated infectious transcripts (FIG. 4). The 5 differences are located at amino acid positions 1414 (Val→Ala); 2718 (Gly→Asp); 2877 (Val→Met); 3228 (Leu→Met); 3278 (Tyr→Glu). The amino acids encoded at these positions by the cDNA sequence which is noninfectious are indicated before the arrow, amino acids at these positions in the copy that is infectious are indicated after the arrow (SEQ ID No. 1). Whether each of the amino acid changes individually will abolish infectivity of the C-strain DNA copy will have to be determined by analysing infectivity of transcripts with individual mutations of each of the five amino acids. However, this finding shows that small differences in the amino acid sequence may be crucial for infectivity of transcripts of a DNA copy of the genome of the C-strain. It also indicates that preparing infectious transcripts of a copy of the sequence of a pestivirus may in practice appear to be impossible because of small differences in sequences (even at the one amino acid level) which may go unnoticed.

C-strain derived mutants that are suitable for (marker) vaccine development are part of the invention. They may contain mutations like deletions, insertions, (multiple) nucleotide mutations, and inserted and/or exchanged genomic fragments originating from other pestivirus strains, in the nucleotide sequence described in SEQ ID No. 1.

The sequence of the C-strain can be divided in four regions suitable for mutation and/or exchange. Region one is the 5′ non-coding sequence running from nucleotides 1 to 373. Region two encodes the structural proteins N^(pro)-C-E2-E3-E1 and runs from nucleotides 374 to 3563. Region three encodes the nonstructural proteins and runs from nucleotides 3564 to 12068. Region four is the 3′ non-coding sequence which runs from nucleotides 12069 to 12311.

One region that is particularly suitable for making C-strain marker vaccines comprises the genomic region encoding the structural proteins N^(pro)-C-E2-E3-E1. This region is located between amino acids 1 and 1063 in the sequence of SEQ ID No. 1. Preferred subregions of this part of the genome are specified by the following amino acid sequences 1-168 (N^(pro)), 169 to 267 (C), 268 to 494 (E2), 495 to 689 (E3), and 690 to 1063 (E1), or parts thereof. As an example the N-terminal antigenic part of the region encoding E1 of the C-strain, running from amino acid 690 to 877, was exchanged with the corresponding region of E1 of strain Brescia (FIG. 4, pPRKflc-h6). The newly generated C-strain derivative is infectious and can be discriminated from the wild-type strain and from strain Brescia through reaction with C-strain and Brescia specific monoclonal antibodies, directed against E1 and E2; as an example, the resulting C-strain reacts with monoclonal antibodies specific for E1 of strain Brescia (Table 1). Thus, the antigenic properties of this new mutant have changed with respect to the parent virus, demonstrating that exchanging the N-terminal half of E1 of the C-strain with that of another CSFV strain is one approach to the development of a C-strain marker vaccine. However, the invention is not restricted to exchange of N-terminal halves of E1 between the C-strain and other CSFV strains. The N-terminal halves of E1 from any other pestivirus strain may be exchanged with corresponding parts of E1 of the C-strain. In this respect, E1 sequences of pestivirus strains which are isolated from pigs, but belong to an antigenic group other than the C-strain, are particularly suitable. Examples of such strains, which were selected on the basis of cross-neutralisation, include strains “Van EE”, “Stam”, “SF UK 87”, “Wisman”, and “5250” (Wensvoort et al. 1989. Vet. Microbiol. 20: 291-306; Wensvoort. 1992. In: Report on meeting of national swine laboratories within the European Community. Jun. 16-17, 1992. VI/4059/92-EN(PVET/EN/1479) 1992, p59-62).

The N-terminal half of E1 has been shown to contain three distinct antigenic domains, A, B and C, located on distinct parts of the E1 protein and each reacting with strongly neutralizing monoclonal antibodies (Wensvoort. 1989. J. Gen. Virol. 70: 2865-2876; Van Rijn et al. 1992. Vet. Microbiol. 33: 221-230; Van Rijn et al. 1993. J. Gen. Virol. 74: 2053-2060). Epitopes conserved among 94 CSFV strains tested, map to domain A, whereas the epitopes of domains B and C are non-conserved (Wensvoort. 1989. J. Gen. Virol. 70: 2865-2876). Mapping of epitopes with hybrids of the E1 genes of strains Brescia and C (Van Rijn et al. 1992. Vet. Microbiol. 33: 221-230), and with deletion mutants of E1 of strain Brescia, suggest that domains A and B+C form two distinct antigenic units in the N-terminal half of E1 (Van Rijn et al. 1993. J. Gen. Virol. 74: 2053-2060). This suggestion was further supported by the finding that the six cysteines located at positions 693, 737, 792, 818, 828, and 856, in the N-terminal half of E1 are critical for the correct folding of E1. However, at least Cys 792 is not crucial for infectivity of strain Brescia, because a monoclonal antibody resistant mutant of this virus was isolated with a Cys→Arg mutation at this position (Van Rijn et al. 1993. Presentation and abstract at the 9th International Congress of Virology, 8-13 August, Glasgow, Scotland).

Whereas small changes in the amino acid sequence may abolish infectivity of the RNA of the C-strain (see Example 2), the cysteine change at position 792 shows that an amino acid change at a position which is less predicted to be suitable for modification without loss of function, may still result in a viable virus mutant. Thus, the effects of a particular arnino acid change on the properties of the virus will have to be determined empirically for each amino acid in the sequence of strain C. This again shows that no obvious target sequences for modification of the C-strain, e.g. for marker vaccine development, can be identified on the basis of previously published information.

Essential to the development of C-strain marker vaccines is the possibility to differentiate serologically between vaccinated pigs and pigs infected with a CSFV field strain. It was shown previously that a live attenuated pseudorabies virus vector expressing E1, or immunoaffinity purified E1, expressed in insect cells with a baculovirus vector, induces a protective immune response in pigs against hog cholera (WO 91/00352; Van Zijl et al. 1991. J. Virol. 65: 2761-2765; Hulst et al. 1993. J. Virol. 67: 5435-5442). It was surprisingly found that mutants of E1 with a deleted A domain or with deleted B+C domains (FIG. 5), also induce a protective immune response in pigs against hog cholera (Table 2). This indicates that protective immunity induced by the vaccine strain does not depend on neutralizing antibodies against both domains A and B+C. Therefore, pestivirus strain mutants having exchanged or mutated only the A domain, or only the B+C domains, or parts thereof, with the corresponding region of another pestivirus, preferably but not exclusively a pestivirus isolated from pigs belonging to a different antigenic group than the C-strain (for examples see above), are also part of the invention. The region of E1 covering domain A and suitable for exchange or mutation, is located between amino acids 785 and 870. Parts of this region may also be suitably exchanged or mutated, e.g. the subregions located between amino acids 785 and 830 and between amino acids 829 and 870. The region of E1 covering domains B+C and suitable for exchange or mutation is located between amino acids 691 and 750. Parts of this region may also be suitably exchanged or mutated, e.g. the subregions located between amino acids 691 and 718 and between amino acids 717 and 750.

Animals infected with pestiviruses develop antibodies against E2 (Kwang et al., 1992. Vet. Microbiol. 32: 281-292; Wensvoort. unpublished observation). Therefore, a second region suitable for (marker) vaccine development via mutation (deletions, insertions, point mutations), or exchange of corresponding genetic material with an antigenically different pestivirus, or with a pestivirus belonging to a different antigenic group, is the region encoding E2.

The C-strain may also be used as a vector for the insertion and expression of heterologous genetic material (sequences). For vector development, heterologous genetic material inserted into the C-strain serves to alter translation strategy of the large ORF and processing of the polyprotein encoded by this ORF. An example of a sequence suitable for altering the translation strategy of the large ORF is a sequence specifying an Internal Ribosome Entry Site (IRES) (Duke et al. 1992. J. Virol. 66: 1602-1609, and references therein). An example of a sequence suitable for altering processing of the polyprotein is a signal sequence responsible for translocation of proteins exported from the cell or inserted into membranes, across the membrane of the endoplasmatic reticulum (Blobel. 1980. Proc. Natl. Acad. Sci. U.S.A. 77: 1496-1500; Kreil. 1981. Annu. Rev. Biochem. 50: 317-348). Signal sequences are cleaved by cellular signal peptidases. However, sequences encoding cleavage sites of viral proteases may as well be used to alter processing of the polyprotein.

Sequences inserted and expressed by a C-strain vector may be used as a marker to identify vaccinated pigs, or may be used to protect pigs against the pathogen from which the heterologous inserted sequence originates. Marker sequences are preferably highly antigenic and belonging to microorganisms not replicating in pigs. They may encode known complete gene products (e.g. capsid or envelope proteins) or antigenic parts of these gene products (e.g. epitopes). Preferably marker sequences originate from viruses belonging to the families: Adenoviridae, Arenaviridae, Arteriviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Rhabdoviridae, Parvoviridae, Poxviridae, Picornaviridae, Reoviridae, Retroviridae, and Togaviridae. However, marker sequences may also encode artificial antigens not normally encountered in nature, or histochemical markers like Escherichia coli β-galactosidase, Drosophila alcohol dehydrogenase, human placental alkaline phosphatase, firefly luciferase, and chloramphenicol acetyltransferase.

Heterologous genetic material encoding one or more proteins inducing protective immunity against disease caused by the pathogen corresponding with the heterologous genetic material may be derived from other pestivirus strains, including sequences of strains specified above, porcine parvovirus, porcine respiratory coronavirus, transmissible gastro-enteritis virus, porcine reproductive and respiratory syndrome virus (Lelystad virus, EP. 92200781.0), Aujeszky's disease virus (pseudorabies virus), porcine endemic diarrhoea virus, and porcine influenza virus, and bacteria, such as Pasteurella multocida, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, Streptococcus suis, Treponema hyodysenteria, Escherichia coli, Leptospira, and mycoplasmata, such as M. hyopneumoniae and M. lyorhinis.

Suitable sites for insertion of heterologous sequences in the C-strain, but not the only ones, are located between amino acid residues 170 and 171, between residues 690 and 691, and between residues 691 and 692 and are indicated in SEQ ID No. 1.

The invention also includes diagnostic tests which can be used to discriminate between pigs vaccinated with a marker vaccine, or a subunit vaccine containing (mutated) E1 and/or (mutated) E2, and pigs infected with a pestivirus field strain. Suitable forms of such differential diagnostic tests are described in Examples 4 and 5. In the conventional non-discrimiminatory CSFV ELISA test, E1 is used as antigen in the complex trapping blocking (CTB) ELISA assay described by Wensvoort et al., 1988. (Vet. Microbiol. 17:129-140). This prior art CTB-ELISA, also called Liquid Phase Blocking ELISA, or double antibody sandwich ELISA, uses two monoclonal antibodies (Mabs) which were raised against E1 of CSFV strain Brescia. The epitope for Mab b3, which is located within domain A, is conserved among CSFV strains, whereas the epitope of Mab b8, which is located within domain C, is nonconserved (Wensvoort. 1989. J. Gen. Virol. 70: 2865-2876). The above CTB-ELISA is sensitive, reliable and specifically detects CSFV specific antibodies in pigs infected with a pestivirus. Thus, the test differentiates between pigs infected with a CSFV strain and pigs infected with e.g. a BVDV strain. However, the test does not differentiate between pigs infected with a CSFV field strain and pigs vaccinated with the C-strain vaccine. Also this test is not suitable in conjunction with an E1 subunit vaccine whether live or dead.

One test according to the invention is a modfied CTB-ELISA, based on only one MAb, e.g. MAb b3. Such a CTB-ELISA, based on only one Mab which Mab is used for binding of the antigen to the surface of an ELISA plate as well as competition with a field serum has not yet been described and is an essential part of this invention. Now that the principle of this test has been described, it can be usefully applied to the development of diagnostic kits for the detection of other antibodies including antibodies against other viruses or other diseases, or antibodies which are indicative for other conditions of the human or animal body. The finding is therefore useful for all CTB-ELISA's, or ELISA's based on the same principle as a CTB-ELISA, which are developed on the basis of a single Mab and a dimerised or multimerised antigen. The claimed test method is also applicable to the determination of other members of pairs of specifically binding partner molecules, such as activators/receptors, enzymes/inhibitors and the like, wherein one of the partners has at least two identical binding sites.

Thus the invention also comprises a method of determining the presence of a test substance (e.g. antibody) capable of specifically binding with a binding site of a binding partner (e.g. antigen), in a sample, by means of competition of said test substance with a measurable amount of a reference substance (antibody) capable of specifically binding with the same binding site of said binding partner, comprising

(1) contacting said sample with (a) said reference substance (antibody) bound to a solid carrier, (b) the binding partner (antigen) of said reference substance, said binding partner molecule containing at least two identical binding sites for said reference substance, and (c) said reference substance (antibody) provided with a label;

(2) measuring the degree of separation of said label from said carrier.

As an example, said binding partner (antigen) to said reference substance (antibody), containing at least two identical binding sites is a dimer of a binding partner (antigen) to said reference substance.

Using the same principle, the invention also comprises a method of determining the presence of a test substance (antigen) having at least two identical binding sites per molecule for specifically binding with a binding partner (antibody), in a sample, comprising

(1) contacting said sample with (a) said binding partner (antibody) bound to a solid carrier, and (b) said binding partner (antibody) provided with a label;

(2) measuring the degree of binding of said label to said carrier.

In these methods, the antibodies and antigens are only referred to by way of example; they may be substituted by other specifically binding partner molecules.

Further provided is a diagnostic kit containing: (a) a reference monoclonal antibody bound to a solid carrier, (b) said reference monoclonal antibody provided with a label; and optionally (c) an antigen to said reference antibody containing at least two identical binding sites for said reference antibody; or a complex between said components (a) and/or (b) and (c); as well as further components for carrying out a competitive immunological assay.

The method is suitable as a differential diagnostic test in conjunction with an E1 subunit vaccine, which has a deletion in one or more epitopes of E1, e.g. domain A. The test is also suitable in conjunction with subunit E1 of which the A domain has been mutated such that antibodies induced against such mutated A domain do not compete with Mab b3 for the epitope of Mab b3. Furthermore, the test is suitable in conjunction with a modified C-strain or other CSFV strain vaccines with a deletion in domain A, with a domain A which has been exchanged with that of a pestivirus belonging to a different antigenic group as CSFV (see above), or with a domain A which has been mutated such that antibodies directed against that domain do not compete with Mab b3 for the epitope of Mab b3. Although the test is described and exemplified for domain A of E1, a similar test based on only Mab b8 can be used in conjunction with a vaccine with a deletion in domains B+C or domain C, with domain B+C or domain C which has been exchanged with that of a pestivirus belonging to a different antigenic group as CSFV (see above), or with domain B+C or domain C which has been mutated such that antibodies directed against those domains do not compete with Mab b8 for the epitope of Mab b8. The test is illustrated in conjunction with Mab b3 or Mab b8 of strain Brescia. However, the test may be usefully set up with other Mabs directed against domain A or domains B+C of E1 of strain Brescia or against domain A or domains B+C of any other CSFV strain, but also with Mabs against analogous domains in E1 of any other pestivirus. The test can also be based on epitopes of E2 (see Example 5). Antigens suitable in the (modified) CTB-ELISAs according to the invention are preferably dimers or multimers of E1 (plus or minus a 3′-TMR) or E2 (see Example 5) of CSFV strains reacting with Mab b3 or Mab b8 or similar MAbs directed against E2 epitopes. In the case of a vaccine with a mutated A domain, dimers or multimers of the antigen used for the diagnostic test may be synthesised by the deletion B+C construct (see Example 5), or in the case of a vaccine with mutated B+C domains, dimers or multimers of the antigen used for the diagnostic test may be synthesised by the deletion A construct (compare FIG. 5 for constructs; compare Examples 4 and 5). The dimerised (or multimerised) form of the E1 antigen is believed to be based on disulphide bridges formed by cysteine residues in the C-terminal part of E1. It allows a very sensitive immunoassay, as the dimerised antigen molecule contains two copies of the epitope of one Mab. Thus, this one Mab can be used for immobilising the dimerised antigen via one epitope, and for labeling the dimerised antigen via the other epitope. Competition by sample serum antibodies raised as a result of field strain infection inhibits binding of the labeled antibody to the antigen, and thus results in a sensitive test for the presence of such antibodies. The invention also relates to diagnostic kits based on this method, which kit comprises E1- or E2-based antigens, and (enzyme-) labeled and immobilised monoclonal antibodies of the same type directed at an E1 or E2 epitope, as well as further conventional components (plates, diluents, enzyme substrate, colouring agents, etc.) for carrying out an immunoassay of the competition type.

The vaccine according to the invention contains a nucleotide sequence as described above, either as such or as a vaccine strain or in a vector or host organism, or a polypeptide as described above, in an amount effective for producing protection against a pestivirus infection. The vaccine can also be a multipurpose vaccine comprising other immunogens or nucleotides encoding these. The vaccines can furthermore contain conventional carriers, adjuvants, solubilizers, emulsifiers, preservatives etc. The vaccines according to the invention can be prepared by conventional methods.

The method of the invention for the production of infectious transcripts of a full-length DNA copy of the genome of a CSFV strain, the C-strain, is useful for any other C-strain derived, or pestivirus strain. The method, described here for a live attenuated CSFV vaccine strain, may also be very usefully applied to in vitro attenuate (modify) the C-strain or any other CSFV or pestivirus strain, for vaccine purposes.

The C-strain vaccine according to the invention allows serological discrimination between vaccinated pigs and pigs infected with a CSFV field strain. Marker vaccines of any other CSFV-strain or pestivirus strain may equally well be obtained using the methods of the invention. Such marker vaccines may be developed for instance by mutating (deletions, point mutations, insertions) the region encoding E1, or the N-terminal half of E1, or domains A or B+C of E1. or the region encoding E2 of the C-strain, or analogous regions in the genomes of C-strain derived, or other pestivirus strains, or by exchanging these regions with the corresponding regions of antigenically different pestiviruses or of pestiviruses belonging to a different antigenic group.

An alternative approach to the development of a C-strain marker vaccine is to add to its genome heterologous genetic material expressing a highly antigenic protein or epitope(s) of a microorganism not replicating in pigs, or of artificial nature and not normally occurring in pigs.

Furthermore such heterologous genetic material may encode antigens inducing protective immunity against a disease caused by a microorganism pathogenic for pigs. Therefore, application of the C-strain, or strains derived from the C-strain, or whatever other pestivirus strain, as a vector for the expression of heterologous antigens inducing protection against a particular disease in a host organism, the host organism being a mammal, is also part of the invention. The construction of recombinant C-strain viruses expressing heterologous sequences and suitable sites for insertion of these heterologous sequences are described above. Analogous recombinant viruses can be made for C-strain derived viruses, or for any other pestivirus. These viruses are therefore also part of the invention.

An essential part of the invention relates to the immunogenic potential of subunit E1 with deletions in domain A, or domains B+C. As summarised in Table 2, both of these mutant E1s are capable of inducing protective immunity in pigs against challenge with a lethal dose of the highly virulent Brescia strain. The use of mutants of E1 containing deletions or other mutations in domains A and B+C as dead subunit vaccine, or as live subunit vaccine expressed by a vector system in the vaccinated animal. against CSF, is also part of the invention. Also mutated E1 together with other antigenic CSFV proteins, e.g. E2 or a mutated form of E2. is suitable as dead or live subunit vaccine (see above).

The invention also includes diagnostic tests which can be used to discriminate between pigs vaccinated with a CSFV marker vaccine, or a subunit vaccine containing (mutated) E1 and/or (mutated) E2, and pigs infected with a pestivirus field strain. Such a diagnostic test may be based on serology, antigen detection, or nucleic acid detection. The choice which test is appropriate in a given case is amongst others dependent on the specificity of the marker used. One suitable form of a serological diagnostic test is the modified CTB-ELISA, described in example 4. According to the invention, this method, based on a CTB-ELISA using a single antibody, is not restricted to the use in the context of CSFV or other pestiviruses, but is also applicable to the determination of other antibodies for other diagnostic purposes in the human or animal field, as well as to the determination of other specifically binding substances.

An example of a suitable antigen detection test in conjunction with a C-strain marker vaccine is a test detecting CSFV field strain E1 and not vaccine strain E1 in the blood of pigs. If the A domain of the C-strain has been modified by e.g. exchange of this domain with that of a pestivirus strain belonging to a different antigenic group than CSFV, such a test may be based on monoclonal antibodies recognizing conserved epitopes of the A domain of CSFV.

However, if the E2 region of the C-strain is modified for marker vaccine development, a serological or antigenic diagnostic test accompanying such a vaccine detects differences between vaccinated and infected animals, in relation to the modified E2 region. Such a diagnostic test thus uses E2 specific sequences as an antigen. These E2 specific sequences may originate from the parent C-strain (see example 5), from CSFV strains which are antigenically different from the C-strain, or from pestiviruses belonging to a different antigenic group than CSFV. However, these E2 specific sequences may also be obtained via mutation (deletion(s), insertion(s), or point mutation(s)) of native E2 of any pestivirus, or may consist of (mutated) parts of E2 of any pestivirus. Dimeric E2 and multimeric E2 may be used as antigen in a diagnostic test (see example 5). Also E2 in conjunction with one monoclonal antibody (oompare Examples 4 and 5) may be used in a CTB-ELISA test, the principle of which has been described above. A diagnostic test based on E2 is described in Example 5. Where an antigen detecting kit is to detect pestivirus E2 and is based on one Mab, such test kist preferably contains an antibody recognising a conserved epitope on E2. Such tests are also part of the invention.

Finally, a diagnostic test may be based on the specific detection of a region of CSFV field strains which is modified in the C-strain. Suitable techniques for this test include nucleic acid hybridisation, e.g. with specific probes, and/or amplification, e.g. with the polymerase chain reaction. Alternatively, C-strain sequences may be distinguished from CSFV field strain sequences by PCR amplification of (a part of the 3′ non-coding region containing the TTTTCTTTTTTTT sequence unique to the C-strain genome.

If the C-strain is modified by insertion of a heterologous marker sequence, any form of a diagnostic test based on this sequence, e.g. based on the antigen, epitope(s), or histochemical product encoded by this sequence, or based on detection of the heterologous genetic information via nucleic acid hybridisation techniques, e.g. specific probes, and/or amplification techniques, like the polymerase chain reaction, is also part of the invention.

EXAMPLE 1 Molecular Cloning and Sequencing of the Genome of the C-strain

Cells and virus Swine kidney cells (SK6-M, EP-A-351901) were grown in Eagle's basal medium containing 5% fetal bovine serum (FBS) and antibiotics. FBS was tested for the presence of BVDV and BVDV antibodies as described (Moormann et al. 1990. Virology 177: 184-198). Only sera free from BVDV and BVDV antibodies were used. The “Chinese” vaccine strain (C-strain) of Classical swine fever virus (CSFV) was adapted to SK6-M cells as described in EP-A-351901. The strain designated “Cedipest” is noncytopathic and was biologically cloned by threefold endpoint dilution. After three amplification steps a cloned virus stock with a titer of 3,5.10⁶ TClD₅₀/ml was produced.

Isolation of Cytoplasmic RNA of SK-6 Cells Infected with the C-strain.

Intracellular RNA from cells infected with the C-strain was isolated essentially as described (Moormann et al. 1990. Virology 177: 184-198). Briefly, monolayers of SK6-M cells in 162 cm² bottles (Costar) were infected with Cedipest at a multiplicity of infection (m.o.i.) of 5 TClD₅₀ per cell. Subsequently, cells were incubated for 1.5 hr at 37° C., and fresh medium was added to a final volume of 40 ml. After 7 hrs Actinomycin D was added to a final concentration of 1 μg/ml. After 24 hrs cells were washed twice with ice cold phosphate buffered saline (PBS), and lysed in ice-cold lysisbuffer (50 mM Tris-HCl pH 82, 0.14 M NaCl, 2 mM MgCl₂, 5 mM DTT, 0.5% [v/v] NP-40, 0.5% [w/v] Na-deoxycholate, and 10 mM vanadyl ribonucleoside complexes (New England Biolabs)). The lysates were centrifuged (4° C., 5 min., 4000 g) and the supernatant was treated with proteinase K (250 μg/ml, final concentration) for 30 min. at 37° C. extracted twice with phenol, chloroform, and isoamyl alcohol (49:49:2), and extracted once with chloroform and isoamyl alcohol (24:1). RNA was stored in ethanol.

Synthesis and Amplification of cDNA

One to two μg of cytoplasmic RNA of cells infected with the C-strain, and 20 pmol (−)sense primer were incubated with 1 μl 10 mM methylmercury hydroxide for 10 min. at room temperature. The denaturated RNA was then incubated with 1 μl 286 mM β-mercaptoethanol for 5 min. at room temperature. The RNA was reverse transcribed with 200-400 units M-MLV reverse transcriptase deficient of RNase H (Promega) for 45 min. at 42° C. in 1×M-MLV reverse transcriptase buffer (50 mM Tris-HCl pH 8.3. 75 mM KCl, 3 mM MgCl₂ and 10 mM DTT), 40 U rRNasin (Promega), and 80 μM of dATP, dGTP, dCTP and dTTP. The final reaction volume was 25 μl. The samples were overlaid with 30 μl of mineral oil (Sigma).

After reverse transcription (RT) the samples were denaturated for 10 min. at 94° C. Portions of 2.5 μl of each RT-reaction were amplified in a polymerase chain reaction (PCR) of 39 cycles (cycle: 94° C., 60 sec.; 55° C., 60 sec. and 72° C., 1-2 min.) in 100 μl Taq polymerase buffer (supplied by the manufacturer of Taq polymerase) containing 1 μM of the (+) as well as the (−) sense primer, 200 μM of each of the four dNTPs, and 2.5 U Taq DNA polymerase (Boehringer Mannheim). The samples were overlaid with 75 μl of mineral oil (Sigma).

Cloning of cDNA Covering the Complete Genome of the C-strain

The genome of the C-strain was cloned indepently twice. During the first round of cloning (FIG. 1A), primers for first strand cDNA synthesis and PCR were selected on the basis of homology between the sequences of the CSFV strains Brescia (Moormann et al. 1990. Virology 177: 184-198) and Alfort (Meyers et al. 1989. Virology 171:555-567), and the BVDV strains Osloss (Renard et al. EP 0208672) and NADL (Collett et al. 1988. Virology. 165: 191-199). The sizes of the cDNA fragments were chosen between 0.5-2.5 kb in order to obtain optimal amplification. Gel purified amplification products were treated with T4 DNA potymerase and Klenow DNA polymerase I, and phosphorylated with T4 polynucleotide kinase. Thereafter, cDNA fragments were ligated with T4 ligase into the Smal site of pGEM4z-blue.

In the second round of cloning (FIG. 1B), primers were selected from the sequence of the cDNA clones obtained after the first round of cloning. Where possible, primers contained restriction sites suitable for cloning of the amplified cDNA fragments. After RT and PCR amplification (see above), cDNA fragments were either cut with two different restriction enzymes, or blunted and phosphorylated (as described above) at one end, and digested with a suitable restriction enzyme at the other end. If it was not possible to use PCR introduced restriction sites located in the primers, a site within the amplified cDNA fragment was chosen for cloning. After gel purification, PCR products were ligated into gel purified pGEM4z-blue (Promega) or pGEM5zf(+) (Promega), digested with restriction enzymes creating ends compatible with those of the PCR products.

To obtain cDNA clones containing the ultimate 5′ and 3′ ends of the genome of the C-strain, we used the 3′-5′ ligation method (Mandl et al. 1991. Journal of Virology 65:4070-4077). Cytoplasmic RNA was isolated from cells infected with the C-strain as described above, and was further purified through a 5.7 CsCl cushion (Moormann and Hulst. 1988. Virus Res. 11: 281-291). Based on results suggesting that there is no Cap structure at the 5′ end of the BVDV genome (Brock et al. 1992. J. Virol. Meth. 38: 39-46), genomic RNA of the C-strain was ligated without previous treatment with pyrophosphatase. Eight μg of RNA was ligated in a reaction mix of 50 mM Tris-HCl pH 8.0, 10 mM MgCl2, 10 mM DTT, 20 U rRNasin (Promega), 10 μg/ml BSA (RNase free) and 1 mM ATP, using 10 U of T4 RNA ligase (New England Biolabs). The mixture was incubated for 4 hrs at 37° C. RNA was extracted with phenol/chloroform, precipitated with ethanol, pelleted, and resuspended in RNase-free water. Portions of 2 μg RNA were reverse transcribed and amplified as described above. Portions of 2 μl of each PCR were reamplified using a nested set of primers. For reverse transcription, a (−)sense primer was used hybridizing to the 5′ noncoding region. For the two PCR amplification steps we used (+)sense primers hybridizing the 3′ noncoding region and (−)sense primers hybridizing to the 5′ noncoding region. After extraction with phenol/chloroform and ethanol precipitation, PCR products were digested with NcoI (incorporated in the (+)sense primer used in the nested PCR) and EagI (nucleotide 81 in the sequence of SEQ ID No. 1), and ligated into the NcoI-EagI sites of pUC21 (Vieira and Messing. 1991. Gene 100: 189-194).

All modification and cloning procedures used in Example 1 were carried out essentially as described (Sambrook et al. 1989. Molecular cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Restriction enzymes and DNA modifying enzymes were commercially purchased and used as described by the suppliers. Plasmids were transformed and maintained in Escherichia coli strain DH5α (Hanahan. 1985. in DNA cloning 1: 109-135).

Sequencing of cDNA Clones.

Plasmid DNA used for sequencing was extracted and purified either by alkaline lysis and LiCl precipitation, or by CsCl centritugation (Sambrook et al. 1989. Molecular loning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). The T7 polymerase based sequencing kit (Pharmacia) was used for direct double stranded sequencing of plasmid DNA. In addition to the SP6, T7, and universal pUC/M13 forward and reverse primers, oligonucleotide primers were used based on the sequence of CSFV strain Brescia (Moormann et al. 1990. Virology 177: 184-198). Primers were synthesised with a Cyclone DNA synthesizer (New Brunswick Scientific) or with a 392 DNA/RNA synthesizer (Applied Biosystems). Sequence reactions were analysed on a 6% acrylamide gel containing 8 M urea. Sequence data were analysed with a Compaq 386 computer using Speedreader hardware, and PCgene software (Intelligenetics Inc, Applied Imaging Corp., Geneva, Switzerland) and with an Apple Macintosh computer using the program MacMollytetra.

Considering the possibility of sequence errors or differences caused by Taq polymerase or heterogeneity of C-strain RNA, the entire genomic sequence of cDNA clones of the C-strain was determined by sequencing a minimum of two cDNA clones, obtained after independent PCR reactions. If differences were observed between the nucleotide sequences of two clones of a particular region, the consensus nucleotide sequence of that region was determined by sequencing a third cDNA clone obtained after another independent PCR reaction (FIG. 1A).

EXAMPLE2 Generation of Infectious Transcripts of 8 Full-length DNA Copy of the Genome of the C-strain

Construction of cDNA Clone pPRKflc-113.

A full-length DNA copy of the genomic RNA of the C-strain was composed according to the scheme depicted in FIG. 3. First, two subciones, one (pPRc64) containing the cDNA sequence of the 5′ half of the genome (nucleotides 1-5,560), and the other (pPRc111) containing the cDNA sequence of the 3′ half of the genome (nucleotides 5,463-12,311) were constructed. Initially construction of the full-length cDNA clone was tried in pGEM4z-blue. However, this approach failed because of instability of the full-length insert in this vector. To increase stability of the clones, inserts of the 5′ and 3′ half clones were recloned in a derivative of the low copy number vector pOK12 (Vieira and Messing. 1991. Gene 100: 189-194), resulting in pPRc108 and pPRc123, respectively. To this end pOK12 was modified by deleting most of the restriction sites of the multiple cloning site (MCS), and the T7 promoter sequence. The resulting vector, pPRK, which was used for all further full-length cloning, still contains unique SpeI, NotI, EagI, BamI, EcoRI, EcoRI, and XbaI sites in the MCS.

In detail, the construction of full-length clone pPRKflc-113 proceeded as follows (FIG. 3). Inserts of plasmids pPRc45 and pPRc46 were joined at the Hpal site, located at nucleotide position 1249 in the sequence of the C-strain (SEQ ID No. 1), resulting in plasmid pPRc49. The insert of pPRc49 was subsequently joined with the insert of pPRc44 at the NslI site located at nucleotide position 3241 (SEQ ID No. 1), resulting in pPRc63. The 5′ half clone pPRc64 (nucleotide 1 to 5560, SEQ ID No. 1) was constructed by joining the insert of pPRc63 with an amplified (PCR) cDNA fragment of the ultimate 5′ region of the genomic RNA of the C-strain as follows. A 5′ end (+)sense primer was synthesised containing an EcoRI and a SalI site followed by the T7 RNA polymerase promoter sequence and the first 23 nucleotides of the genomic RNA of the C-strain. This primer and a (−) sense primer of the second round of cloning were used to amplify a cDNA fragment that was digested with EcoRI and XhoI cloned into EcoRI-XhoI (nucleotide 216 in SEQ ID No. 1) digested pPRc63. Finally, the insert of pPRc64 was recloned into EcoRI-XbaI digested pPRK resulting in pPRc108.

The 3′ half clone pPRc111 (nucleotide 5,463 to 12,311, SEQ ID No. 1) was constructed by joining 4 second round clones (pPRc67, 53, 58, and 55) and one first round clone (pPRc14). The inserts of pPRc67 and pPRc53 were joined at the NheI site located at nucleotide position 7,778, resulting in pPRc71. The inserts of pPRc55 and pPRc58 were joined at the ApaI site located at nucleotide position 10,387, resulting in pPRc65. The inserts of pPRc65 and pPRc14 were subsequently joined at the AflII site at nucleotide position 11,717, resulting in pPRc73. The insert of pPRc73 was joined with the insert of pPRc71 at the PstI site located at nucleotide position 8,675, resulting in pPRc79. Then, the insert of pPRc79, which contains the complete 3′ terminal sequence of the cDNA of the C-strain, was modified such that an SrlI site was introduced which after digestion generated the exact 3′ end of the C-strain cDNA sequence (for exact run-off transcription at the 3′end). To achieve this, a 3′ end (−)sense primer was synthesised containing an SrlI and an XbaI site and 18 nucleotides complementary to the 3′ terminal sequence of the genomic RNA of the C-strain. This primer and a (+)sense primer of the first round of cloning were used to amplify a cDNA fragment. This fragment was digested with SpeI (nucleotide position 11,866, SEQ ID No. 1) and XbaI and cloned into SpeI-XbaI digested pPRc79, resulting in pPRc111.

Full-length cDNA clone pPRKflc-113, finally, was constructed by inserting the C-strain specific NcoI⁵⁵³²-XbaI^(mcs) fragment of pPRc111 into NcoI⁵⁵³²-XbaI^(mcs) digested pPRc108.

Construction of Full-length Clone pPRKflc-133.

Full-length cDNA clone pPRKflc-113 still had, besides silent nucleotide mutations, 5 point mutations leading to amino acid changes compared to the amino acid sequence determined from the sequence of at least two first round cDNA clones. These 5 point mutations in pPRKflc-1 13 were changed to the predominant sequence (2 out of 3) through exchange of affected DNA fragments with corresponding DNA fragments containing the predominant sequence.

The 5′ half cDNA clone pPRc108, with a point mutation at nucleotide position 4,516, was changed by replacing the ScaI³⁴¹³-NcoI⁵⁵³² fragment of pPRc108 with that of pPRc124. Clone pPRc124 was made by exchanging the PvuII⁴⁴⁸⁵-NheI⁵⁰⁶⁵ fragment of pPRc44 by the corresponding fragment of pPRc32 (compare FIG. 1). The new 5′ half cDNA clone was designated pPRc129.

For cloning purposes a 3′ half clone was constructed by deleting the 5′ part of the C-strain sequence of pPRKflc-113 from the SalI site in the vector (compare FIG. 3) up to the Hpal site at nucleotide position 5,509 (SEQ ID No. 1), resulting in pPRc123. In pPRc123 mutations at nucleotide positions 8,526, 9,002, 10,055, and 10,205 had to be changed. The mutation at position 8,526 was restored in two steps. First, the ApaI^(8,506)-PstI^(8,675) fragment of pPRc53 was exchanged with that of pPRc90, resulting in pPRc125. Second, the NheI^(7,378)-PstI^(8,675) fragment of pPRc123 was exchanged with that of pPRc125, resulting in pPRc127.

To be able to restore the 3 mutations at positions 9,002, 10,055, and 10,205, we first modified pPRc58 such that the FspI site in the vector was deleted. To this end the EcoRI^(mcs)-NdeI fragment of pPRc58 was deleted (NdeI cuts in pGEM4z-blue), resulting in pPRc126. Plasmid pPRc126 was used for restoring the mutations at positions 10,055 and 10,205 by replacing its SacI^(9,975)-ApaI^(10,387) fragment with the corresponding fragment of pPRc96, resulting in pPRc128. The mutation at position 9002 was restored by replacing the AatII-FspI⁹⁰¹⁶ (AatII cuts in pGEM4z-blue) of pPRc128 with the AatII-FspI^(9,016) fragment of pPRc90, resulting in pPRc130. Finally, the PstI^(8,675)-ApaI^(10,387) fragment of pPRc127 was replaced with the corresponding fragment of pPRC130, resultng in plasmid pPRc132. All subcloning steps in which single mutations were changed were verified by sequencing.

Full-length clone pPRKflc-133 was constructed by inserting the NcoI^(5,532)-XbaI^(mcs) fragment of pPRc132 into NcoI^(5,532)XbaI^(mcs) digested pPRc129.

Construction of a Hybrid Full-length Clone pPRKflc-h6.

Antigenically different but viable C-strain mutants can be made from pPRKflc-133, by exchanging part of the E1 gene of this construct with that of CSFV strain Brescia. To this end, the NheI^(2,443)-AflIII^(2,999) fragment of pPRc129 was replaced with the corresponding fragment of pPEh6 (van Rijn et al., 1992), resulting in the 5′ half hybrid clone pPRc139. Hybrid full-length clone pPRKflc-h6 was constructed by inserting the NcoI^(5,532)-XbaI^(mcs) fragment of pPRc132 into pPRc139. This clone now contained the antigenic region of E1 of CSFV-strain Brescia including a unique BgIII site.

All modification and cloning procedures used in Example 2 were carried out essentially as described (Sambrook et al. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Restriction enzymes and DNA modifying enzymes were commercially purchased and used as described by the suppliers. Plasmids were transformed and maintained in Escherichia coli strain DH5α (Hanahan. 1985. in DNA cloning 1: 109-135).

In vitro RNA Transcription

Plasmid DNA used for in vitro RNA transcription was purified using Qiagen columns (Westburg), according to manufacturers conditions. After linearisation with XbaI or SrfI, plasmid DNA was extracted with phenol and chloroform, precipitated with ethanol, vacuum dried and dissolved in an appropriate volume of RNase-free water.

One μg of linearised plasmid DNA was used as template for in vitro transcription. RNA was synthesised at 37° C. for 1 hr in 100 μl reaction mixtures containing 40 mM Tris-HCl pH 7.5, 6 mM MgCl₂, 2 mM Spermidine, 10 mM DTT, 100 U rRNasin (Promega), 0.5 mM each of ATP, GTP, CTP, UTP and 170 Units of T7 RNA polymerase (Pharmacia). Template DNA was removed by digestion with RNase-free DNaseI (Pharmacia) for 15 min. at 37° C., followed by extraction with phenol and chloroform, and ethanol precipitation. The RNA was dissolved in 20 μl RNase-free H₂O, and quantitated by UV measurement at 260 nm.

RNA Transfection

The RNA transfection mix was composed by gently mixing 50 μl of a lipofectine (Gibco BRL) dilution (10 μg lipofectine in RNase-free H₂O) and 50 μl of an RNA solution (1 μg RNA in RNase-free H₂O), and incubation of this mix at room temperature for 15 minutes. Subconfluent monolayers of SK6-cells in φ35 mm, 6-well tissue culture plates (Greiner) were used for RNA transfection. The cells were washed twice with Dulbecco's modified Eagles medium (DMEM). Then 1 ml of DMEM was added to the cells, followed by the RNA transfection mix. After incubation for 16 hrs at 37° C., the medium was replaced by 2 ml DMEM supplemented with 5% FBS. Incubation was continued for another 3 days at 37° C. Then cells were immunostained with CSFV specific monoclonal antibodies (Mabs) by the immunoperoxidase monolayer assay (IPMA) as described by Wensvoort et al. (Vet. Microbiol. 1986. 12: 101-108).

Characterisation of Recombinant C-strain Viruses.

The supernatants of transfected cells were brought on confluent monolayers of SK6-cells in wells of φ35 mm, and incubated for 5 days at 37° C. Cells of the transfected monolayers were trypsinised and diluted 7.5 times with DMEM and grown for another 7 days at 37° C. in 75 cm² flasks (Costar). Hereafter, virus stocks were prepared by freeze-thawing the cells twice, clarifying cell suspensions by centrifugation at 5,000×g for 10 min. at 4° C., and harvesting of the supernatants.

Virus was characterised by IPMA, and by restriction analysis of RT-PCR amplified viral fragments. After infection of SK6-cells with viruses FLc-h6 and Flc-133, monolayers were incubated for 4 days at 37° C. Subsequently, monolayers were immunostained using Mabs directed against conserved (Mab b3, domain A) and non-conserved (Mabs b5 and b6, domains B+C) epitopes on E1 of Brescia, and with Mabs specific for the C-strain and directed against E1 (Mab c2) or E2 (Mab c5) (Wensvoort, G. 1989. In Thesis, pp 99-113, Utrecht, The Netherlands). Monolayers of SK6-cells infected with native Brescia virus or native C-strain virus were controls in this assay. The results are presented in Table 1, and are as expected. Briefly, Mab b3 recognizes an epitope on E1 conserved among CSFV strains, and therefore recognizes all strains in Table 1. Mabs b5 and b6 do not recognize E1 of the C-strain and thus only react with strains Brescia and Flc-h6. In contrast, Mabc2 does not recognize E1 of strain Brescia. and thus only reacts with strains C and FLc-133. Finally, Mab c5 does not recognize E2 of strain Brescia, and therefore reacts with all viruses in Table 1 except strain Brescia.

The genomic RNA of virus FLc-h6 should contain a unique BglII site, which is located in the E1 gene (see above). To check for the presence of this site, cytoplasmic RNA was isolated from SK6-cells infected with recombinant virus FLc-h6, or infected with FLc-133, PCR-amplified as described above, using primers described by Van Rijn et al, 1993. J. Gen. Virol. 74:2053-2060), and digested with BglII. Indeed, the amplified fragment of 1,091 basepairs of FLc-h6 was cut by BglII, resulting in fragments of 590 and 501 basepairs, whereas the amplified fragment of FLc-133 remained intact.

EXAMPLE 3 Immunisation of Pigs with Deletion Mutants of E1

Construction and Expression of Deletion Mutants of E1 of CSFV Strain Brescia.

It was previously shown that TMR-less E1 of CSFV strain Brescia, expressed by insect cells, induces a protective immune response in pigs against CSF (Huist et al., 1993. J. Virol. 67: 5435-5442). Two distinct antigenic units, A and B+C, in the N-terminal half of E1, which induce neutralizing antibodies against CSFV, were also defined (Wensvoort. 1989. J. Gen. Virol. 70:2865-2876; Van Rijn et al. 1992. Vet. Microbiol. 33: 221-230; Van Rijn et al. 1993. J. Gen. Virol. 74: 2053-2060). Moreover, neutralizing antibodies directed against domain A and domains B+C act synergistically in neutralizing CSFV (Wensvoort. 1989. J. Gen. Virol. 70: 2865-2876). To evaluate the immunogenicity of mutant E1s with a deletion of domains B+C or with a deletion of domain A, relevant constructs in a baculovirus vector were made, and expressed mutant proteins were tested in pigs.

Baculoviruses expressing mutant E1s were constructed via overlap recombination of wild type AcNPV (Autographa californica nuclear polyhedrosis virus) DNA and DNA of transfer vector pAcMo8 containing the sequence encoding a particular mutant E1. Transfer vector pAcMo8 was derived from pAcAs3 (Vlak et al., 1990. Virology 179: 312-320) by inserting a T directly 5′ of the first base (G) of the unique BamH1 site of the latter vector. In this way an ATG start codon was generated overlapping the first G of the BamHI site. Messenger RNA is transcribed from heterologous sequences inserted into the BamHI site by the AcNPV p10 promoter.

The sequences encoding mutant E1s were derived from the E1 insert of pPRb2 (Van Rijn et al., 1992. Vet. Microbiol. 33: 221-230) via PCR amplification. To this end two primers were constructed which contained a BamHI site in their sequence. The 5′ end (+ sense) primer has the sequence 5′-AGA TTG GAT CCT AAA GTA TTA AGA GGA CAG GT-3′ (SEQ ID No. 10). The underlined sequence in this primer is identical to nucleotides 2362-2381 in the sequence of strain Brescia (Moormann et al., 1990. Virology 177: 184-198), bold letters indicate the BamHI site. The 3′ end (− sense) primer contains a stop-codon adjacent to the BamH1 site. It has the sequence 5′-TA GTC GGA TCC TTA GAA TTC TGC GAA GTA ATC TGA-3′ (SEQ ID No. 11). The underlined sequence in this primer is complementary to nucleotides 3433-3453 in the sequence of strain Brescia (Moormann et al., 1990. Virology 177: 184-198); bold letters indicate the BamHI site, and letters in italics indicate the stop-codon.

Amplified sequences were cloned into the BamHI site of pAcMo8 and checked for a correct orientation in the vector by restriction enzyme analysis. The correct transfer vector was designated pPAb11. Overlap recombination between AcNPV DNA and DNA of pPAb11, and selection and purification of a baculovirus vector expressing E1 was performed as described (Hulst et al., 1993. J. Virol. 67: 5435-5442). Further characterisation of E1 in radioimmunoprecipitation assays, and with E1 specific Mabs was also described by Hulst et al. (J. Virol., 1993. 67: 5435-5442). The resulting recombinant baculovirus expresses wild-type Brescia E1 without a TMR (compare 2^(nd) bar from top in FIG. 3). This TMR-less E1 is secreted from the cells (Hulst et al., 1993. J. Virol. 67: 5435-5442).

Deletion of the region encoding domains B+C from the E1 gene of pPAb11 was achieved by exchanging the NheI-BglII fragment of this construct with the corresponding fragment of pPEh14 (Van Rijn et al., 1993. J. Gen. Virol. 74: 2053-2060). The resulting transfer vector was designated pPAb16. It contains a deletion in the E1 gene running from codon 693 to 746. Similarly, the region encoding domain A was deleted from pPAb11 by exchanging the NheI-BglII fragment of pPAb11 with the corresponding fragment of pPEh18 (Van Rijn et al., 1993. J. Gen. Virol. 74: 2053-2060), resulting in transfer vector pPAb12. pPAb12 contains a deletion in the E1 gene running from codon 800 to 864.

Recombinant baculoviruses expressing the deleted E1s were constructed, selected, and characterised with regard to their E1 expression products, as described above.

Immunisation and Challenge Exposure of Pigs.

Groups of four (or two) specific-pathogen-free (SPF), 6 to 8 week old pigs were vaccinated intramuscularly on day 0 with 1 ml of a double water-oil emulsion containing 4 μg (mutant) E1, and revaccinated on day 28 with 1 ml of a double water-oil emulsion containing 15 μg (mutant) E1 (Table 2). The construction of mutant E1 containing a deletion in domain A, or a deletion in domain B/C, and of wild type E1 is described above and specified by the constructs depicted in FIG. 5. For the first vaccination on day 0, supernatant of insect cells infected with the appropriate recombinant baculoviruses was used. The amount of E1 in the supernatant was calibrated as described before (Hulst et al., 1993. J. Virol. 67: 5435-5442). For revaccination on day 28, E1 was immunoaffinity-purified from the supernatant of the infected insect cells (Hulst et al., 1993. ibid). Pigs of all vaccinated groups, and an unvaccinated control group of two SPF animals, were challenged intranasally with 100 LD₅₀ of CSFV strain Brescia 456610 (Terpstra and Wensvoort. 1988. Vet. Microbiol. 16: 123-128). This challenge dose leads to acute disease in unprotected pigs characterised by high fever and thrombocytopenia starting at days 3 to 5 and to death at days 7 to 11. Heparinised (EDTA) blood samples were taken on days 40, 42, 45, 47, 49, 51, 53, and 56 after vaccination, and analysed for thrombocytes and CSFV virus as described (Hulst et al., 1993. ibid). Serum blood samples were taken on days 0, 21, 28, 42, and 56 and tested in the CTB-ELISA (Wensvoort et al., 1988. Vet. Microbiol. 17: 129-140) and in the neutralizing peroxidase-linked assay (NPLA, Terpstra et al. 1984. Vet. Microbiol. 9: 113-120), to detect (neutralizing) antibodies against CSFV. Test results in the CTB-ELISA are expressed as the percentage inhibition of a standard signal; <30% inhibition is negative, 30-50% inhibition is doubtful, >50% inhibition is positive. NPLA titers are expressed as the reciprocal of the serum dilution that neutralised 100 TCID₅₀ of strain Brescia in 50% of the replicate cultures.

All animals were observed daily for signs of disease, and body temperatures were measured. Clinical signs of disease were: Fever, anorexia, leukopenia, thrombocytopenia, and paralysis.

EXAMPLE 4 Development of a CTB-ELISA (CTB-DIF) for CSFV, Based on One Monoclonal Antibody

Description of the Diagnostic Test.

This example describes a CTB-ELISA (CTB-DIF), which is a modification of the existing CTB-ELISA (Wensvoort et al., 1988. Vet. Microbiol. 17: 129-140) for the detection of CSFV specific antibodies.

The CTB-DIF is based on the finding that SF21 cells infected with a recombinant baculovirus expressing E1-TMR, efficiently secrete dimerised E1 into the medium. This dimerised secreted E1 was detected when media of cells infected with the above baculovirus was analysed on western blot after electrophoresis in SDS-PAGE under non-reduced conditions. For E1 specific Mabs, two copies of an epitope are present on dimers of E1 (one on each monomer). Thus, in conjunction with the dimerised antigen a particular E1 specific Mab can be used as capture antibody, coated to the wall of a microtiter plate well, as well as detecting, horseradish peroxidase (HRPO) conjugated antibody.

The CTB-DIF is shown to be useful in conjunction with an E1 subunit vaccine which has a deletion in domain A (see FIG. 5 for construct) and is shown to distinguish between CSFV specific antibodies induced in pigs vaccinated with E1 with a deleted domain A. and CSFV specific antibodies induced in pigs infected with low-virulent CSFV strains Henken, Zoelen, Bergen, 331, and Cedipest (EP-A-351901).

Four SPF pigs, numbered 766, 786, 789, and 770, were vaccinated with mutant E1 containing a deletion in domain A. as described in example 3 (see also Table 2), and challenged with virulent CSFV strain Brescia on day 44 after vaccination. Sera taken on days 28, 42, and 56 after vaccination, were tested.

Sera against the low-virulent CSFV strains were also prepared in groups of four SPF pigs. Sera from pigs infected with strains Henken, Zoelen, Bergen, and 331 were tested at days 0, 21, 28 and 42 after infection. Sera from pigs vaccinated with the Cedipest vaccine were tested at days 0, 44, 72, and 170 after vaccination.

Three different serological tests were performed with the above sera. Test 1 is the neutralizing peroxidase-linked assay (NPLA) described by Terpstra et al. 1984. (Vet. Microbiol. 9: 113-120), to detect neutralizing antibodies against CSFV. Test 2 is the CTB-ELISA (Wensvoort et al., 1988. Vet. Microbiol. 17: 129-140), to routinely detect antibodies against CSFV.

The CTB-DIF uses Mab b3 (also known as CVI-HCV-39.5) (Wensvoort. 1989. J. Gen. Virol. 70:2865-2876.), which recognizes an epitope located in domain A1 of E1 of CSFV. The wells of an ELISA plate are coated with Mab b3 (dilution 1:2.000) (capture antibody). After the wells are washed, Mab b3, conjugated to (HRPO), (dilution 1:4.000) (detecting antibody), is added to the wells. Media of Sf21 cells infected with a baculovirus producing E1-TMR and containing dimerised E1 to a concentration of 20 μg/ml is diluted 1:500, and pre-incubated with the test serum (diluted 1:2.5). The serum-antigen mixture is then added to the conjugate in the wells of the coated ELISA plate. After incubation, the wells are washed again and the chromogen-substrate solution is added. If both the capture and conjugated Mab have bound to the antigen, the HRPO induces a chromogenic reaction, indicating that the test serum is negative for CSFV antibodies. If the epitope on the antigen is blocked by antibodies from the test serum, the HRPO-conjugate will be washed away and the wells will remain clear, indicating the test serum contains antibodies against CSFV, domain A1. The results with the three different serological tests are indicated in Table 3.

Sera of pigs vaccinated with E1 with a deletion in domain A1. do react in the NPLA and CTB-ELISA, and not in the CTB-DIF, on day 42 after vaccination. After challenge with virulent CSFV strain Brescia sera of the same pigs react positively in all the 3 tests on day 56 after vaccination (day 12 after challenge), indicating that a booster response has taken place after challenge. Starting on day 21 after infection, sera from pigs vaccinated with strains Henken, Zoelen, Bergen, and 331 react positively in the NPLA, the CTB-ELISA, and the CTB-DIF. Starting on day 44 after vaccination, the same holds true for pigs vaccinated with the Cedipest vaccine strain.

Thus, the CTB-DIF exactly performs as desired, and is suited to accompany a CSFV marker vaccine with a mutated domain A of E1, such that antibodies directed against this mutated domain A do not compete with Mab b3 for the epitope of Mab b3.

The antigen used in the CTB-DIF is the dimerised TMR-less wild type Brescia E1 depicted in FIG. 5. However, dimerised E1 synthesised by the “deletion domains B+C” construct of FIG. 5 is also suitable as an antigen in the test.

EXAMPLE 5 Comparison of CTB-ELISA's for CSFV Based on E1 and E2

Description of the Diagnostic Tests

This example describes a modification of the CTB-DIF of example 4, and a CTB-ELISA based on E2 of CSFV, and compares the sensitivity of these ELISAs with 3 other CTB-ELISAs detecting antibodies directed against E1 and the NPLA (Terpstra et al. 1984. Vet. Microbiol. 9: 113-120).

The CTB-DIF of example 4, called E1-Bac-DIF in Tables 4 to 8, uses intact TMR-less E1 synthesized in insect cells (SF21 cells) as an antigen. The modification of E1-Bac-DIF, called E1-Bac-dBC-DIF, uses TMR-less E1 synthesized in insect cells (SF 21 cells) with a deleted domain B+C (compare FIG. 5) as an antigen. As established on western blot, TMR-less E1 with deleted domains B+C is secreted from the cell as a dimer (results not shown). Test E1-bac-dBC-DIF is performed as follows. The wells of an ELISA plate are coated with Mab b3 (dilution 1:4,000) (capture antibody), 16 h at 37 ° C., and washed. Medium containing dimerised antigen E1-dBC to a concentration of 20 μg/ml is diluted 1:50, and pre-incubated with the test serum (dilution 1-2.5) (0.5 h at 37° C.). The serum-antigen mixture is then added to the coated ELISA plate. After incubation, 1 h at 37° C., the wells are washed and Mab b3, conjugated to HRPO (dilution 1:1,000) (detection antibody), is added. After incubation, 1 h at 37° C., the wells are washed again and the chromogen-substrate solution is added. The chromogenic reaction is performed for 10 minutes at room temperature. The interpretation of the chromogenic reaction is the same as explained in example 4.

Other CTB-ELISAs detecting antibodies directed against E1 of CSFV described in Tables 4 to 8 are the E1-CSFV ELISA. using native E1 from CSFV infected cells as antigen (Wensvoort et al., 1988. Vet. Microbiol. 17:129-140); the E1-Bac and E1-Bac-DIF ELISAs, use TMR-less E1 synthesized in insect cells as antigen. The E1-CSFV and E1-Bac ELISAs use CSFV Mabs b3 and b8 (Wensvoort 1989. J. Gen. Virol. 70: 2,865-2,876) as capture and detection antibody, respectively, whereas the E1-Bac-DIF ELISA uses only Mab b3 as both capture and detection antibody. The E1-CSFV ELISA is performed exactly as described by Wensvoort et al., 1988. (Vet. Microbiol. 17:129-140). The E1-Bac, and E1-Bac-DIF ELISAs are performed as described above for the E1-Bac-dBC-DIF with the following modifications. In the E1-Bac ELISA the antigen used is a 1:400 dilution of dimerized E1 present in the medium of SF21 cells, infected with the relevant E1 baculovirus construct (compare FIG. 5), at a concentration of 20 μg/ml. Mab b8 which is conjugated to HRPO is the detection antibody in this ELISA, and is used at a dilution of 1:1000. The E1-Bac-DIF ELISA uses the same antigen as the E1-Bac ELISA but at a dilution of 1:200. HRPO conjugated Mab b3 is used as detection antibody in this ELISA at a dilution of 1:1,000.

The E2-Bac ELISA uses CSFV E2 antigen synthesized in SF21 cells infected with the Bac CE2 construct (Hulst et al., 1994. Virology 200: 558-565). Because E2 is not secreted from the infected insect cells, the lysate of these cells is used. Like E1, most of E2 is found as dimerized molecules when lysates of infected cells are analyzed under non-denaturing conditions in SDS-PAGE gels (results not shown). The CTB-ELISA developed on the basis of this E2 antigen performs optimally in conjunction with Mabs C5 and C12 (Wensvoort, G. 1989. In Thesis, pp99-113, Utrecht). However, also E2 in conjunction with only Mab C5 or Mab C12 may be used. In a competition assay Mabs C5 and C12 inhibit each other with regard to binding to E2. This indicates that these Mabs recognize the same, or overlapping epitopes on E2 (results not shown). The E2-Bac ELISA is performed as follows. Mab C12 is diluted 1:1,000, and coated to the wells of an ELISA plate (16 h at 37° C.). Hereafter, wells are washed. Lysates of SF21 cells infected with Bac CE2, diluted 1:1,250, are preincubated with the test serum (1:1) for 0.5 h at 37° C. The serum-antigen mixture is then added to the wells of the coated plates and incubated for 1 h at 37° C. Subsequently plates are washed and incubated with Mab C5 conjugated to HRPO (dilution 1:2,000). After 1 h at 37° C. plates are washed again and the chromogen-substrate solution is added. The chromogenic reaction is performed for 10 minutes at room temperature. The interpretation of the chromogenic reaction is the same as explained in example 4. All above described dilutions are performed in NPLA buffer+4% PS (Terpstra et al., Vet. Microbiol. 9: 113-120).

Table 4 shows the results of the analysis of sera of 3 SPF pigs vaccinated with the Cedipest vaccine with the above described CTB-ELISAs and the NPLA. Sera were analyzed at days 0, 16, 23, 30, 37, 44, 50, 72, 113, 141, and 170 after vaccination. Tables 5 to 8 show the results of the analysis with the above described CTB-ELISAs and the NPLA of sera of groups of 5 SPF pigs infected with the low-virulent CSFV strains 331, Bergen, Henken, and Zoelen, respectively. Sera were analyzed at days 0, 10, 14, 17, 24, 28, 35, and 42 after infection. Starting at day 16 after vaccination, sera from pigs vaccinated with the Cedipest strain react in each of the 5 CTB-ELISAs as well as in the NPLA. At this time point the sensitivity of the E2-Bac ELISA and the E1-Bac-dBC-DIF is as good, if not better, than that of the other 3 CTB-ELISAs. From day 37 after vaccination up till day 170, all sera react consistently (positive) in the 5 CTB-ELISAs as well as the NPLA. Sera of pigs infected with the low-virulent CSFV strains also react in all 5 CTB-ELISAs as well as in the NPLA. With an occasional exception consistency in the reaction of the sera in the 5 CTB-ELISAs and the NPLA is observed from day 21 after infection up till day 42. More sera of animals infected with low virulent strains need to be analyzed to be able to conclude whether there are significant differences between the sensitivity of the 5 CTB-ELISAs early after infection (up till day 17).

It can be concluded that the E2-Bac ELISA and the E1-Bac-CTB-DIF ELISA both perform as desired. Therefore the E2-Bac ELISA is suitable to accompany a CSFV marker vaccine (eg. subunit E1, whether mutated or not, a C-strain marker vaccine modified in the E2 region) which does not induce antibodies that compete with the Mabs in this ELISA. The E1-Bac-dBC-DIF ELISA is as suitable as the E1-Bac-DIF ELISA (CTB-DIF ELISA of example 4) to accompany a CSFV marker vaccine with a mutated domain A of E1, such that antibodies directed against this mutated domain A do not compete with Mab b3 for the epitope of Mab b3.

Description of the Figures

FIG. 1.

Schematic representation of the cDNA clones used to determine the nucleotide sequence of the C-strain. FIG. 1A indicates the first round cDNA clones (see text). cDNA clones with numbers 32, 90, and 96 were used to change pPRKflc-113 into pPRKflc-133 (see example 2). Clone 14 was the only first round cDNA clone used for construction of pPRKflc-113 (see FIG. 3). FIG. 1B indicates second round cDNA clones (see text). The numbered second round cDNA clones were used to construct pPRKflc-1 13 (see SEQ ID No. 1). Positions of the cDNA with respect to the nucleotide sequence of the genome of the C-strain are indicated by the scale bar (in kilobases) at the bottom of the figure. A schematic representation of the currently identified genes of CSFV, and their organisation in the CSFV genome is indicated at the top of the figure.

There is no consensus yet among workers in the field about the nomenclature of pestivirus proteins. The E2 protein as described here is also called gp42 (Tamura et al. 1993. Virology 193: 1-10), gp44/48 (Thiel et al. 1991. J. Virol. 65: 4705-4712) or E0 (R{umlaut over (u)}menapf et al. 1993. J. Virol. 67: 3288-3294). The E3 protein is also called gp25 (Tamura et al. 1993. Virology 193: 1-10), gp33 (Thiel et al. 1991. J. Virol. 65: 4705-4712) or E1 (R{umlaut over (u)}menapf et al. 1993. J. Virol. 67: 3288-3294). The E1 protein of this invention is also called gp53 (Tamura et al. 1993. Virology 193: 1-10), gp55 (Thiel et al. 1991. J. Virol. 65:4705-4712), gp51-54 (Moormann et al. 1990. Virology 177:184-198) and E2 (R{umlaut over (u)}menapf et al. 1993. J. Virol. 67: 3288-3294). The N-terminal autoprotease N^(pro) of CSFV (p20 of BVDV, Wiskerchen et al. 1991. J. Virol. 64: 4508-4514), also called p23, was identified by Thiel et al. 1991. (J. virol. 65: 4705-4712). Cleavage of the recognition sequence, which is conserved among pestiviruses, of this protease results in the N-terminus of C (Stark et al. 1993. J. Virol. 67: 7088-7095).

FIGS. 2A-2B.

Alignment of the nucleotide sequences of the 5′ (A) and 3′ (B) non-coding regions of CSFV strains Brescia, Alfort, and C. Except for the first 12 nucleotides, the 5′ non-coding sequence of strain Brescia has been described by Moormann et al., 1990. Virology 177: 184-198. The first 12 nucleotides of the 5′ non-coding region of strain Brescia have not been published before. Like the ultimate 5′ and 3′ sequences of the genome of the C-strain, they were determined with the 3′-5′ RNA ligation method described in Example 1 of this patent application. Except for the first 9 nucleotides, the 5′ non-coding sequence of strain Alfort has been described by Meyers et al., 1989. Virology 171: 555-567. The first 9 nucleotides of the genome of strain Alfort were published by Meyers in a Thesis entitled: “Virus der Klassischen Schweinepest: Genomanalyse und Vergleich mit dem Virus der Bovinen Viralen Diarrhöe”. 1990. T{umlaut over (u)}bingen, Germany. The sequences of the 3′ non-coding regions of strains Brescia and Alfort have been described by Moormann et al., 1990. Virology 177: 184-198 and Meyers et al., 1989. Virology 171: 555-567, respectively. The ATG start codon and the TGA stopcodon of the large ORF (compare SEQ ID No. 1), are underlined.

FIG. 3.

Construction scheme of full-length cDNA clone pPRKflc-113. Clone numbers have been explained in the legend of FIG. 1. Fusion sites of inserts of clones are indicated by vertical lines. The sites corresponding with these lines are indicated at the bottom of the figure. Underlined clone numbers indicate cDNA clones having pOK12 (Vieira and Messing. 1991. Gene 100: 189-194) derived vector sequences (see FIG. 4). The 5′ and 3′ ends of pPRKflc-113 were tailor made via PCR amplification of cDNA fragments (see text Example 2). The amplified fragments are indicated with PCR. The scale bar at the bottom of the figure, and the schematic representation of the genome organisation of CSFV, have been described in the legend of FIG. 1.

FIG. 4.

Schematic representation of the vector sequences and full-length cDNA inserts in clones pPRKflc-113, pPRKflc-133, and pPRKflc-h6. The construction of vector pPRK, a derivative of pOK12 (Vieira and Messing. 1991. Gene 100: 189-194), has been described in Example 2. Kan^(R), kanamycin resistance gene; ORI, origin of replication: 'i, gene encoding repressor of β-galactosidase gene; PO, promoter/operator region of β-galactosidase gene; lacZ, part of the β-galactosidase gene encoding the ∝ subunit of β-galactosidase. Several restriction sites of the vector, and the sequences directly flanking the full-length inserts in the vector, are indicated. Relevant sites have been described in the text of Example 2. The lollypops and numbers in pPRKflc-113 correspond to the nucleotides of the five codons which were changed in this construct, resulting in pPRKflc-133. The latter construct has the sequence as indicated in SEQ ID No. 1.

The black box in pPRKflc-h6 indicates the region of E1 of pPRKflc-133 that was exchanged with the corresponding region of strain Brescia. Whether transcripts derived from a particular full-length construct are infectious (+) or not (−) is indicated to the right of the construct. T7, T7 promoter sequence. Inserts of full-length constructs are indicated in relation to a scale bar (in kilobases) representing the nucleotide sequence of the C-strain as indicated in SEQ ID No. 1.

FIG. 5.

Schematic representation of mutant E1 proteins expressed in insect cells with a baculovirus vector. All E1 proteins are encoded by the nucleotide sequence of strain Brescia (Moormann et al., 1990. Virology 177: 184-198), and start at their N-terminus with the Lys at codon position 668 in the large ORF of this sequence. The C-terminus of native E1 is the Leu at codon position 1,063 in the large ORF, whereas the C-termini of the three other E1 proteins are located at amino acid position 1,031. The dotted boxes in the bars represent the N-terrninal signal sequence, running from amino acid residues 668 to 689, the internal hydrophobic sequence, running from amino acid residues 806 to 826, and the C-terminal transmembrane region (TMR), located in the region running from amino acid residues 1,032 to 1,063, of E1. The deleted amino acid sequences in mutant E1s with a deleted B+C or A domain are indicated by interruptions in the bars representing these proteins. The location of these deletions in relation to the amino acid sequence of E1 can be determined from the scale bar at the bottom of the figure. The scale bar indicates the location of E1 in the amino acid sequence encoded by the large ORF of strain Brescia.

TABLE 1 Characterisation of recombinant C-strain viruses Mabs specific for CSFV directed against E1 directed against E2 Brescia “C” “C” conserved specific specific specific virus epitopes epitopes epitopes epitopes “C” + − + + Brescia + + − − FLc-133 + − + + FLc-h6 + + − +

TABLE 2 Vaccination of pigs with deletion mutants of E1 % Inhibition in the Results of CSFV Construct CTB-ELISA on days: Neutralizing antibody titer on days: challenge: Pig no. used 0 21 28 42 56 0 21 28 42 56 Disease Viremia Death 766 Deletion 0 12 0 78 99 <12.5 <25 <25 3,200 >3,200 −−−−− − − 768 Domain 0 0 0 74 99 <12.5 <25 <25 2,400 >3,200 −−−−− − − 769 A 0 16 0 99 99 <12.5 <25 <25 2,400 >3,200 −−−−− − − 770 0 14 0 83 99 <12.5 <25 <25 400 >3,200 −−−−− − − 771 Deletion 0 36 25 100 99 <12.5 <25 <25 300 >3,200 −−−−− − − 772 Domain 0 25 29 100 99 <12.5 <25 <25 1,200 >3,200 −−−−− − − 773 B + C 0 7 0 100 99 <12.5 <25 <25 200 150 −−−−− − − 774 0 49 52 99 99 <12.5 <25 <25 300 >3,200 −−−−− − − 792 Wild type 0 35 31 98 100 <12.5 2,000 1,200 >3,200 >3,200 +−−−− − − 794 BresciaE1 0 48 40 98 100 <12.5 <25 <25 1,600 >3,200 +−−−− − − 775 None 0 2 0 0 <12.5 <25 <25 <25 +++++ + + 795 0 0 0 0 <12.5 <25 <25 <25 +++++ + + Groups of four (or two) SPF pigs were inoculated intramuscularly on day 0 with 4 μg and on day 28 with 15 μg modified E1 protein. On day 42, the pigs were challenged intranasally with 100 LD50 of CSF strain Brescia 456610. All animals were observed daily for signs of disease. Blood samples were taken at days 0, 21, 28, 42, and 56 and tested in the CTB-ELISA and the neutralisation test for the detection of antibodies against CSFV #(Terpstra et al., 1984. Vet. Microbiol. 9: 113-120). Clinical signs of disease were: Fever, anorexia, leukopenia, thrombocytopenia, and paralysis and the presence or absence of these signs is indicated in that order by a + or − in the table listed under disease.

TABLE 3 Differential diagnostic ELISA test for CSFV Serum DPV or DPI^(a) NPLA^(b) CTB-ELISA^(c) CTB-DIF^(c) 766 28 <25 9 27 768 28 <25 0 27 769 28 <25 17 0 770 28 <25 11 0 766 42 3200 81 0 768 42 2400 52 0 769 42 2400 99 26 770 42 400 65 0 766 56 >3200 99 65 768 56 >3200 100 104 769 56 >3200 101 105 770 56 >3200 101 104 Henken 0 <12.5 5 18 Henken 21 50 76 47 Henken 28 75 92 88 Henken 42 300 100 102 Zoelen 0 <12.5 0 0 Zoelen 17 37.5 85 71 Zoelen 21 150 90 80 Zoelen 42 400 100 108 Bergen 0 <12.5 1 49 Bergen 21 25 96 100 Bergen 28 100 99 95 Bergen 42 300 100 103 331 0 18.75 4 15 331 21 100 92 90 331 28 300 99 99 331 42 300 100 105 Cedipest 0 <12.5 0 19 Cedipest 44 75 85 93 Cedipest 72 50 89 102 Cedipest 170 150 98 106 ^(a)DPV: days post vaccination; DPI: days post infection ^(b)NPLA titers are expressed as the reciprocal of the serum dilution neutralizing 100 TCID₅₀ of HCV strain Brescia in 50% of the replicate cultures (Terpstra et al. 1984. Vet Microbiol. 9: 113-120). ^(c)Complex trapping blocking-ELISA, CTB-ELISA, or differential CTB-ELISA, CTB-DIF. Test results are expressed as the percentage inhibition of a standard signal; <30% inhibition is negative, 30-50% inhibition is doubtful, >50% inhibition is positive.

TABLE 4 Comparison of CTB-ELISA's with CSFV strain Cedipest sera DPV^(a) CTB-ELISA^(c) or E1 E1- E1-Bac E1-Bac E2- strain pig DPI NPLA^(b) CSFV Bac DIF dBC-DIF Bac Cedi- 1 0 <12.5 21 ND ND ND 0 pest 2 0 <12.5 28 8 0 0 0 3 0 <12.5 25 0 0 0 0 1 16 25 60 0 56 62 79 2 16 25 66 51 26 76 79 3 16 19 11 15 0 11 62 1 23 25 54 66 60 68 79 2 23 50 81 57 54 75 74 3 23 25 25 37 32 54 74 1 30 50 76 87 80 81 75 2 30 75 87 ND ND ND ND 3 30 19 60 40 28 57 82 1 37 50 82 90 80 87 85 2 37 50 87 84 61 85 85 3 37 19 49 62 63 79 77 1 44 75 84 94 99 92 88 2 44 75 90 89 74 93 92 3 44 25 66 68 79 93 90 1 50 75 86 93 92 98 89 2 50 150 91 95 96 97 91 3 50 19 74 67 58 95 88 1 72 50 86 92 94 99 81 2 72 200 94 96 93 100 89 3 72 25 53 76 66 99 73 1 113 75 94 99 100 100 92 2 113 200 94 98 100 99 89 3 113 75 93 99 100 96 84 1 141 75 91 100 91 100 89 2 141 150 76 100 95 100 85 3 141 50 87 94 95 100 85 1 190 150 92 97 100 100 94 2 170 150 85 97 100 100 86 3 170 150 74 88 84 98 88 ^(a,b)For explanation see Table 3 footnotes a and b, respectively. ^(c)CTB-ELISA's E1CSFV, E1-Bac, E1-Bac-DIF, E1-Bac-dBC-DIF and E2-Bac are explained on Example 5. Test results are expressed as the percentage inhibition of a standard signal; <30% inhibition is negative, 30-50% inhibition is doubtful, >50% inhibition is positive.

TABLE 5 Comparison of CTB-ELISA's with CSFV strain 331 sera CTB-ELISA^(c) E1 E1- E1-Bac E1-Bac E2- strain pig DPI^(a) NPLA^(b) CSFV Bac DIF dBC-DIF Bac 331 1 0 <12.5 0 0 0 8 0 2 0 <12.5 4 5 0 0 0 3 0 <19 4 13 0 0 0 4 0 <12.5 0 14 0 0 0 5 0 <12.5 5 11 11 0 0 1 10 <12.5 0 13 0 14 13 2 10 <12.5 0 11 0 0 13 3 10 <12.5 0 20 16 24 31 4 10 <12.5 0 29 0 9 26 5 10 <12.5 0 24 7 38 18 1 14 <12.5 0 34 9 34 8 2 14 <12.5 2 18 0 0 36 3 14 19 28 67 22 60 60 4 14 19 35 77 37 60 10 5 14 25 32 99 74 90 23 1 17 19 7 84 53 69 0 2 17 <12.5 23 0 0 0 4 3 17 25 63 93 62 87 54 4 17 37 55 82 36 80 0 5 17 37 69 100 84 94 3 1 21 37 57 84 100 50 39 2 21 37 29 52 0 26 3 3 21 100 76 93 100 96 96 4 21 50 76 90 100 91 63 5 21 75 65 ND ND ND 72 1 28 75 73 95 100 96 96 2 28 25 59 89 100 79 58 3 28 300 78 100 100 100 100 4 28 150 74 93 100 80 82 5 28 100 72 98 100 100 91 1 35 75 83 95 100 97 98 2 35 150 80 98 100 100 96 3 35 300 80 99 100 100 100 4 35 200 81 ND ND ND ND 5 35 150 82 98 100 100 90 1 42 150 81 98 100 96 99 2 42 200 80 100 94 100 98 3 42 300 79 94 100 100 100 4 42 200 79 97 100 99 100 5 42 150 80 98 100 100 90 ^(a,b,c)See footnotes of Table 4.

TABLE 6 Comparison of CTB-ELISA's with CSFV strain Bergen sera CTB-ELISA^(c) E1 E1- E1-Bac E1-Bac E2- strain pig DPI^(a) NPLA^(b) CSFV Bac DIF dBC-DIF Bac Ber- 1 0 <12.5 0 2 0 0 0 gen 2 0 <12.5 0 0 0 0 0 3 0 <12.5 0 2 0 0 0 4 0 <12.5 0 7 0 0 0 5 0 <12.5 0 4 0 2 0 1 10 <12.5 0 0 6 0 27 2 10 <12.5 0 5 0 0 4 3 10 12.5 0 25 0 27 21 4 10 <12.5 0 15 0 13 29 5 10 12.5 0 14 0 6 21 1 14 <12.5 0 51 32 18 12 2 14 <12.5 8 12 9 10 4 3 14 37 20 76 53 72 11 4 14 12.5 0 55 8 77 18 5 14 <12.5 0 17 9 10 0 1 17 25 57 93 84 73 3 2 17 <12.5 28 45 0 51 29 3 17 75 75 100 78 90 0 4 17 37 47 88 57 85 16 5 17 12.5 23 54 11 55 0 1 21 25 76 96 100 100 96 2 21 19 62 78 54 72 67 3 21 50 77 ND ND ND 63 4 21 50 72 95 100 93 96 5 21 50 51 80 97 88 0 1 28 100 81 100 100 100 96 2 28 37 80 97 100 100 81 3 28 100 80 96 100 100 72 4 28 50 81 100 100 100 100 5 28 150 79 98 100 99 3 1 35 150 84 98 100 100 100 2 35 50 82 95 100 100 49 3 35 100 79 100 100 100 66 4 35 200 79 100 100 100 82 5 35 200 79 98 100 100 21 1 42 300 82 100 97 89 74 2 42 300 81 98 100 100 49 3 42 300 82 100 65 100 65 4 42 200 81 98 92 100 74 5 42 600 81 98 100 98 0 ^(a,b,c)See footnotes of Table 4.

TABLE 7 Comparison of CTB-ELISA's with CSFV strain Henken sera CTB-ELISA^(c) E1 E1- E1-Bac E1-Bac E2- strain pig DPI^(a) NPLA^(b) CSFV Bac DIF dBC-DIF Bac Hen- 1 0 <12.5 0 0 0 0 0 ken 2 0 <12.5 0 0 0 0 0 3 0 <12.5 1 0 0 0 0 4 0 <12.5 0 2 0 0 0 5 0 <12.5 0 0 0 0 0 1 10 <12.5 0 0 0 0 0 2 10 <12.5 3 6 1 1 25 3 10 <12.5 0 5 12 0 52 4 10 <12.5 0 6 0 4 27 5 10 <12.5 0 12 0 0 8 1 14 <12.5 0 0 6 1 0 2 14 <12.5 0 54 22 29 10 3 14 50 5 57 67 100 20 4 14 <12.5 0 7 0 34 0 5 14 <12.5 0 12 10 9 0 1 17 <12.5 0 0 0 7 0 2 17 12.5 48 73 26 63 53 3 17 75 75 100 94 100 35 4 17 19 7 56 0 60 23 5 17 12.5 0 29 0 16 15 1 21 <12.5 29 0 0 0 0 2 21 50 75 ND ND ND ND 3 21 300 84 ND ND ND ND 4 21 19 36 68 76 78 34 5 21 50 63 83 61 82 32 1 28 <12.5 0 0 0 0 0 2 28 75 80 92 100 100 92 3 28 600 80 99 100 100 82 4 28 50 58 93 100 100 100 5 28 50 79 99 100 100 100 1 35 <12.5 22 13 13 0 1 2 35 200 78 95 100 100 82 3 35 300 78 100 100 100 75 4 35 75 75 98 100 100 100 5 35 150 80 98 100 100 97 1 42 <12.5 17 12 9 0 0 2 42 400 79 ND ND ND ND 3 42 400 79 ND ND ND ND 4 42 400 79 98 100 100 100 5 42 300 82 98 100 100 90 ^(a,b,c)See footnotes of Table 4.

TABLE 8 Comparison of CTB-ELISA's with CSFV strain Zoelen sera CTB-ELISA^(c) E1 E1- E1-Bac E1-Bac E2- strain pig DPI^(a) NPLA^(b) CSFV Bac DIF dBC-DIF Bac Zoe- 1 0 <12.5 0 0 0 0 0 len 2 0 <12.5 0 5 0 0 0 3 0 <12.5 14 4 0 0 0 4 0 19 6 0 0 0 0 5 0 <12.5 16 35 0 19 0 1 10 <12.5 0 16 8 3 31 2 10 <12.5 0 14 0 0 15 3 10 <12.5 0 10 2 8 24 4 10 19 12 8 0 0 22 5 10 <12.5 0 27 27 12 24 1 14 19 19 60 18 75 41 2 14 <12.5 4 36 4 34 10 3 14 <12.5 19 26 14 23 12 4 14 25 26 91 40 92 39 5 14 12.5 0 50 16 41 0 1 17 37 61 96 76 91 64 2 17 19 29 94 62 73 0 3 17 12.5 23 41 16 45 4 4 17 37 65 97 82 95 0 5 17 37 48 90 60 75 0 1 21 150 78 95 100 99 84 2 21 19 68 89 100 94 58 3 21 37 60 73 99 77 0 4 21 75 75 92 100 95 46 5 21 37 54 ND ND ND 57 1 28 200 75 100 100 100 100 2 28 150 76 100 100 100 89 3 28 75 77 97 100 100 56 4 28 300 79 97 100 100 84 5 28 100 67 100 100 100 80 1 35 400 82 100 100 100 100 2 35 150 72 100 100 100 91 3 35 200 81 98 100 100 60 4 35 150 80 94 100 100 77 5 35 100 79 99 100 100 89 1 42 400 82 93 100 100 100 2 42 200 67 99 100 100 78 3 42 400 83 94 100 100 84 4 42 300 82 99 100 100 86 5 42 150 82 94 100 100 32 ^(a,b,c)See footnotes of Table 4.

11 12311 base pairs nucleic acid single linear cDNA unknown CDS 374..12067 1 GTATACGAGG TTAGTTCATT CTCGTATACA CGATTGGACA AATCAAAATT ATAATTTGGT 60 TCAGGGCCTC CCTCCAGCGA CGGCCGAACT GGGCTAGCCA TGCCCATAGT AGGACTAGCA 120 AAACGGAGGG ACTAGCCATA GTGGCGAGCT CCCTGGGTGG TCTAAGTCCT GAGTACAGGA 180 CAGTCGTCAG TAGTTCGACG TGAGCAGAAG CCCACCTCGA GATGCTACGT GGACGAGGGC 240 ATGCCAAGAC ACACCTTAAC CCTAGCGGGG GTCGCTAGGG TGAAATCACA CCACGTGATG 300 GGAGTACGAC CTGATAGGGC GCTGCAGAGG CCCACTATTA GGCTAGTATA AAAATCTCTG 360 CTGTACATGG CAC ATG GAG TTG AAT CAC TTT GAA CTT TTA TAC AAA ACA 409 Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr 1 5 10 AAC AAA CAA AAA CCA ATG GGA GTG GAG GAA CCG GTG TAC GAT GCC ACG 457 Asn Lys Gln Lys Pro Met Gly Val Glu Glu Pro Val Tyr Asp Ala Thr 15 20 25 GGG AGA CCG TTG TTC GGA GAC CCG AGT GAG GTA CAC CCA CAA TCA ACA 505 Gly Arg Pro Leu Phe Gly Asp Pro Ser Glu Val His Pro Gln Ser Thr 30 35 40 CTG AAG CTA CCA CAT GAT AGG GGT AGA GGC AAC ATT AAA ACA ACA CTG 553 Leu Lys Leu Pro His Asp Arg Gly Arg Gly Asn Ile Lys Thr Thr Leu 45 50 55 60 AAG AAC CTA CCT AGG AAA GGC GAC TGC AGG AGC GGC AAC CAT CTA GGC 601 Lys Asn Leu Pro Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly 65 70 75 CCG GTC AGT GGG ATA TAT GTA AAA CCC GGC CCT GTC TTT TAC CAG GAC 649 Pro Val Ser Gly Ile Tyr Val Lys Pro Gly Pro Val Phe Tyr Gln Asp 80 85 90 TAC ATG GGC CCG GTC TAC CAT AGA GCC CCT CTG GAG TTT TTT GAC GAA 697 Tyr Met Gly Pro Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu 95 100 105 GTG CAG TTC TGC GAG GTG ACC AAA AGG ATA GGT AGG GTG ACA GGT AGC 745 Val Gln Phe Cys Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser 110 115 120 GAC GGA AAG CTT TAC CAT ACA TAT GTG TGC ATC GAT GGC TGC ATA CTG 793 Asp Gly Lys Leu Tyr His Thr Tyr Val Cys Ile Asp Gly Cys Ile Leu 125 130 135 140 CTG AAG CTG GCC AAG AGG GGT GAG CCA AGA ACC CTG AAG TGG ATT AGA 841 Leu Lys Leu Ala Lys Arg Gly Glu Pro Arg Thr Leu Lys Trp Ile Arg 145 150 155 AAT TTC ACC GAC TGT CCA TTG TGG GTT ACC AGT TGC TCC GAT GAT GGC 889 Asn Phe Thr Asp Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly 160 165 170 GCA AGT GGG AGT AAA GAG AAG AAG CCA GAT AGG ATC AAC AAA GGC AAA 937 Ala Ser Gly Ser Lys Glu Lys Lys Pro Asp Arg Ile Asn Lys Gly Lys 175 180 185 TTA AAA ATA GCC CCA AAA GAG CAT GAG AAG GAC AGC AGA ACT AGG CCA 985 Leu Lys Ile Ala Pro Lys Glu His Glu Lys Asp Ser Arg Thr Arg Pro 190 195 200 CCT GAC GCT ACG ATC GTG GTG GAA GGA GTA AAA TAC CAG GTC AAA AAG 1033 Pro Asp Ala Thr Ile Val Val Glu Gly Val Lys Tyr Gln Val Lys Lys 205 210 215 220 AAA GGT AAA GTT AAA GGA AAG AAT ACC CAA GAC GGC CTG TAC CAC AAC 1081 Lys Gly Lys Val Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn 225 230 235 AAG AAT AAA CCA CCA GAA TCT AGG AAG AAA CTA GAA AAA GCC CTA TTG 1129 Lys Asn Lys Pro Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu 240 245 250 GCA TGG GCG GTG ATA GCA ATT ATG TTG TAC CAA CCA GTT GAA GCC GAA 1177 Ala Trp Ala Val Ile Ala Ile Met Leu Tyr Gln Pro Val Glu Ala Glu 255 260 265 AAT ATA ACT CAA TGG AAC CTG AGT GAC AAC GGC ACT AAT GGT ATC CAG 1225 Asn Ile Thr Gln Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln 270 275 280 CAT GCT ATG TAC CTT AGA GGG GTT AAC AGA AGC TTG CAT GGG ATC TGG 1273 His Ala Met Tyr Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp 285 290 295 300 CCG GGG GAA ATA TGC AAA GGA GTC CCA ACC CAC CTG GCC ACA GAC GTG 1321 Pro Gly Glu Ile Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Val 305 310 315 GAG CTG AAA GAA ATA CAG GGA ATG ATG GAT GCC AGC GAG GGG ACA AAC 1369 Glu Leu Lys Glu Ile Gln Gly Met Met Asp Ala Ser Glu Gly Thr Asn 320 325 330 TAT ACG TGC TGT AAG TTA CAG AGA CAT GAA TGG AAC AAA CAT GGA TGG 1417 Tyr Thr Cys Cys Lys Leu Gln Arg His Glu Trp Asn Lys His Gly Trp 335 340 345 TGT AAC TGG CAC AAT ATA GAC CCC TGG ATA CAG CTG ATG AAT AGA ACC 1465 Cys Asn Trp His Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr 350 355 360 CAA GCA GAC TTG GCA GAA GGC CCT CCG GTC AAG GAG TGC GCT GTG ACT 1513 Gln Ala Asp Leu Ala Glu Gly Pro Pro Val Lys Glu Cys Ala Val Thr 365 370 375 380 TGC AGG TAC GAT AAA GAT GCT GAC ATC AAC GTG GTT ACC CAG GCT AGA 1561 Cys Arg Tyr Asp Lys Asp Ala Asp Ile Asn Val Val Thr Gln Ala Arg 385 390 395 AAC AGG CCA ACA ACC CTG ACC GGC TGC AAG AAA GGG AAA AAT TTT TCT 1609 Asn Arg Pro Thr Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser 400 405 410 TTT GCG GGT ACA GTT ATA GAG AGC CCA TGT AAT TTC AAT GTT TCC GTG 1657 Phe Ala Gly Thr Val Ile Glu Ser Pro Cys Asn Phe Asn Val Ser Val 415 420 425 GAG GAT ACC TTG TAT GGG GAT CAT GAG TGC GGC AGT TTG CTC CAG GAC 1705 Glu Asp Thr Leu Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp 430 435 440 GCA GCT CTG TAC CTA GTA GAT GGA ATG ACC AAC ACT ATA GAG AAT GCC 1753 Ala Ala Leu Tyr Leu Val Asp Gly Met Thr Asn Thr Ile Glu Asn Ala 445 450 455 460 AGA CAG GGA GCA GCG AGG GTG ACA TCC TGG CTC GGG AGG CAA CTC AGA 1801 Arg Gln Gly Ala Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Arg 465 470 475 ACT GCT GGG AAG AGG TTG GAG GGT AGA AGC AAA ACC TGG TTT GGC GCT 1849 Thr Ala Gly Lys Arg Leu Glu Gly Arg Ser Lys Thr Trp Phe Gly Ala 480 485 490 TAT GCC CTA TCG CCT TAC TGT AAT GTA ACA AGC AAA ATA GGG TAC ATA 1897 Tyr Ala Leu Ser Pro Tyr Cys Asn Val Thr Ser Lys Ile Gly Tyr Ile 495 500 505 TGG TAC ACT AAC AAC TGC ACC CCA GCT TGC CTC CCC AAA AAC ACA AAG 1945 Trp Tyr Thr Asn Asn Cys Thr Pro Ala Cys Leu Pro Lys Asn Thr Lys 510 515 520 ATA ATA GGC CCT GGT AAA TTT GAC ACT AAT GCA GAA GAC GGA AAG ATT 1993 Ile Ile Gly Pro Gly Lys Phe Asp Thr Asn Ala Glu Asp Gly Lys Ile 525 530 535 540 CTC CAT GAG ATG GGG GGC CAC CTA TCA GAA TTT CTG CTG CTT TCT CTG 2041 Leu His Glu Met Gly Gly His Leu Ser Glu Phe Leu Leu Leu Ser Leu 545 550 555 GTT GTT CTG TCT GAC TTC GCC CCT GAA ACA GCC AGC GCG TTA TAC CTC 2089 Val Val Leu Ser Asp Phe Ala Pro Glu Thr Ala Ser Ala Leu Tyr Leu 560 565 570 ATT TTG CAC TAC GTG ATT CCT CAA CCC CAT GAT GAA CCT GAA GGC TGC 2137 Ile Leu His Tyr Val Ile Pro Gln Pro His Asp Glu Pro Glu Gly Cys 575 580 585 GAT ACG AAC CAG CTG AAT CTA ACA GTA GAA CTC AGG ACT GAA GAC GTA 2185 Asp Thr Asn Gln Leu Asn Leu Thr Val Glu Leu Arg Thr Glu Asp Val 590 595 600 ATA CCG TCA TCA GTC TGG AAT GTT GGT AAA TAT GTG TGT GTT AGA CCA 2233 Ile Pro Ser Ser Val Trp Asn Val Gly Lys Tyr Val Cys Val Arg Pro 605 610 615 620 GAC TGG TGG CCA TAT GAA ACC GAG GTG GCT CTG TTA TTT GAA GAG GTA 2281 Asp Trp Trp Pro Tyr Glu Thr Glu Val Ala Leu Leu Phe Glu Glu Val 625 630 635 GGA CAG GTC GTA AAG TTA GCC TTA CGG GCG CTG AGG GAT TTG ACT AGG 2329 Gly Gln Val Val Lys Leu Ala Leu Arg Ala Leu Arg Asp Leu Thr Arg 640 645 650 GTC TGG AAT AGC GCA TCA ACC ATT GCA TTC CTC ATC TGC TTG ATA AAA 2377 Val Trp Asn Ser Ala Ser Thr Ile Ala Phe Leu Ile Cys Leu Ile Lys 655 660 665 GTA TTA AGA GGA CAG ATC GTG CAA GGT GTG GTA TGG CTG TTA CTA GTA 2425 Val Leu Arg Gly Gln Ile Val Gln Gly Val Val Trp Leu Leu Leu Val 670 675 680 ACT GGG GCA CAA GGC CGG CTA GCC TGC AAG GAA GAT TAC AGG TAC GCA 2473 Thr Gly Ala Gln Gly Arg Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala 685 690 695 700 ATA TCG TCA ACC GAT GAG ATA GGG CTA CTT GGG GCC GGA GGT CTC ACC 2521 Ile Ser Ser Thr Asp Glu Ile Gly Leu Leu Gly Ala Gly Gly Leu Thr 705 710 715 ACC ACC TGG AAG GAA TAC AAC CAC GAT TTG CAA CTG AAT GAC GGG ACC 2569 Thr Thr Trp Lys Glu Tyr Asn His Asp Leu Gln Leu Asn Asp Gly Thr 720 725 730 GTT AAG GCC AGT TGC GTG GCA GGT TCC TTT AAA GTC ACA GCA CTT AAT 2617 Val Lys Ala Ser Cys Val Ala Gly Ser Phe Lys Val Thr Ala Leu Asn 735 740 745 GTG GTC AGT AGG AGG TAT TTG GCA TCA TTG CAT AAG AAG GCT TTA CCC 2665 Val Val Ser Arg Arg Tyr Leu Ala Ser Leu His Lys Lys Ala Leu Pro 750 755 760 ACT TCC GTG ACA TTC GAG CTC CTG TTC GAC GGG ACC AAC CCA TCA ACT 2713 Thr Ser Val Thr Phe Glu Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr 765 770 775 780 GAG GAA ATG GGA GAT GAC TTC AGG TCC GGG CTG TGC CCG TTT GAT ACG 2761 Glu Glu Met Gly Asp Asp Phe Arg Ser Gly Leu Cys Pro Phe Asp Thr 785 790 795 AGT CCT GTT GTT AAG GGA AAG TAC AAT ACG ACC TTG TTG AAC GGT AGT 2809 Ser Pro Val Val Lys Gly Lys Tyr Asn Thr Thr Leu Leu Asn Gly Ser 800 805 810 GCT TTC TAT CTT GTC TGC CCA ATA GGG TGG ACG GGT GTC ATA GAG TGC 2857 Ala Phe Tyr Leu Val Cys Pro Ile Gly Trp Thr Gly Val Ile Glu Cys 815 820 825 ACA GCA GTG AGC CCA ACA ACT CTG AGA ACA GAA GTG GTA AAG ACC TTC 2905 Thr Ala Val Ser Pro Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe 830 835 840 AGG AGA GAC AAG CCC TTT CCG CAC AGA ATG GAT TGT GTG ACC ACC ACA 2953 Arg Arg Asp Lys Pro Phe Pro His Arg Met Asp Cys Val Thr Thr Thr 845 850 855 860 GTG GAA AAT GAA GAT TTA TTC TAT TGT AAG TTG GGG GGC AAC TGG ACA 3001 Val Glu Asn Glu Asp Leu Phe Tyr Cys Lys Leu Gly Gly Asn Trp Thr 865 870 875 TGT GTG AAA GGC GAG CCA GTG GTC TAC ACA GGG GGG CTA GTA AAA CAA 3049 Cys Val Lys Gly Glu Pro Val Val Tyr Thr Gly Gly Leu Val Lys Gln 880 885 890 TGT AGA TGG TGT GGC TTC GAC TTC GAT GGG CCT GAC GGA CTC CCG CAT 3097 Cys Arg Trp Cys Gly Phe Asp Phe Asp Gly Pro Asp Gly Leu Pro His 895 900 905 TAC CCC ATA GGT AAG TGC ATT TTG GCA AAT GAG ACA GGT TAC AGA ATA 3145 Tyr Pro Ile Gly Lys Cys Ile Leu Ala Asn Glu Thr Gly Tyr Arg Ile 910 915 920 GTA GAT TCA ACG GAC TGT AAC AGA GAT GGC GTT GTA ATC AGC ACA GAG 3193 Val Asp Ser Thr Asp Cys Asn Arg Asp Gly Val Val Ile Ser Thr Glu 925 930 935 940 GGG AGT CAT GAG TGC TTG ATC GGT AAC ACG ACT GTC AAG GTG CAT GCA 3241 Gly Ser His Glu Cys Leu Ile Gly Asn Thr Thr Val Lys Val His Ala 945 950 955 TCA GAT GAA AGA TTG GGC CCT ATG CCA TGC AGA CCT AAA GAG ATT GTC 3289 Ser Asp Glu Arg Leu Gly Pro Met Pro Cys Arg Pro Lys Glu Ile Val 960 965 970 TCT AGT GCT GGA CCT GTA AAG AAA ACC TCC TGT ACA TTC AAC TAC ACA 3337 Ser Ser Ala Gly Pro Val Lys Lys Thr Ser Cys Thr Phe Asn Tyr Thr 975 980 985 AAA ACT TTG AAG AAC AGG TAC TAT GAG CCC AGG GAC AGC TAC TTC CAG 3385 Lys Thr Leu Lys Asn Arg Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe Gln 990 995 1000 CAA TAT ATG CTT AAG GGT GAG TAT CAG TAC TGG TTT GAC CTG GAT GCG 3433 Gln Tyr Met Leu Lys Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Asp Ala 1005 1010 1015 1020 ACT GAC CGC CAC TCA GAT TAC TTC GCA GAA TTT GTT GTC TTG GTG GTG 3481 Thr Asp Arg His Ser Asp Tyr Phe Ala Glu Phe Val Val Leu Val Val 1025 1030 1035 GTA GCA CTG TTA GGA GGA AGA TAT GTC CTG TGG CTG ATA GTG ACC TAC 3529 Val Ala Leu Leu Gly Gly Arg Tyr Val Leu Trp Leu Ile Val Thr Tyr 1040 1045 1050 GTA GTT CTA ACA GAA CAA CTC GCC GCT GGT TTA CCA TTG GGC CAG GGT 3577 Val Val Leu Thr Glu Gln Leu Ala Ala Gly Leu Pro Leu Gly Gln Gly 1055 1060 1065 GAG GTA GTG TTG ATA GGG AAC TTA ATT ACC CAT ACA GAC ATT GAG GTC 3625 Glu Val Val Leu Ile Gly Asn Leu Ile Thr His Thr Asp Ile Glu Val 1070 1075 1080 GTA GTA TAT TTT TTA CTA CTC TAT TTG GTC ATG AGG GAT GAA CCT ATA 3673 Val Val Tyr Phe Leu Leu Leu Tyr Leu Val Met Arg Asp Glu Pro Ile 1085 1090 1095 1100 AAG AAA TGG ATA CTG CTG CTG TTC CAT GCT ATG ACT AAC AAT CCA GTC 3721 Lys Lys Trp Ile Leu Leu Leu Phe His Ala Met Thr Asn Asn Pro Val 1105 1110 1115 AAG ACC ATA ACA GTG GCA TTG CTC ATG GTT AGT GGA GTT GCC AAG GGT 3769 Lys Thr Ile Thr Val Ala Leu Leu Met Val Ser Gly Val Ala Lys Gly 1120 1125 1130 GGA AAG ATA GAT GGC GGT TGG CAG CGA CTG CCG GGG ACC AGC TTT GAC 3817 Gly Lys Ile Asp Gly Gly Trp Gln Arg Leu Pro Gly Thr Ser Phe Asp 1135 1140 1145 ATC CAA CTC GCG CTG ACA GTT ATA GTA GTC GCT GTG ATG TTG CTG GCA 3865 Ile Gln Leu Ala Leu Thr Val Ile Val Val Ala Val Met Leu Leu Ala 1150 1155 1160 AAG AGA GAT CCG ACT ACT GTC CCC TTG GTT ATA ACA GTG GCA CCC CTG 3913 Lys Arg Asp Pro Thr Thr Val Pro Leu Val Ile Thr Val Ala Pro Leu 1165 1170 1175 1180 AGG ACA GCT AAG ATG ACT AAC GGA CTT AGT ACG GAT ATA GCC ATA GCC 3961 Arg Thr Ala Lys Met Thr Asn Gly Leu Ser Thr Asp Ile Ala Ile Ala 1185 1190 1195 ACA GTG TCA GCA GCG TTG CTA ACC TGG ACC TAC ATT AGT GAC TAT TAC 4009 Thr Val Ser Ala Ala Leu Leu Thr Trp Thr Tyr Ile Ser Asp Tyr Tyr 1200 1205 1210 AGA TAC AAG ACC TGG CTA CAG TAC CTT ATC AGC ACA GTG ACA GGT ATC 4057 Arg Tyr Lys Thr Trp Leu Gln Tyr Leu Ile Ser Thr Val Thr Gly Ile 1215 1220 1225 TTT TTA ATA AGG GTA CTG AAG GGA ATA GGT GAG TTG GAT TTA CAC ACT 4105 Phe Leu Ile Arg Val Leu Lys Gly Ile Gly Glu Leu Asp Leu His Thr 1230 1235 1240 CCG ACC TTG CCA TCT CAT AGA CCC CTC TTT TTC ATT CTC GTG TAC CTT 4153 Pro Thr Leu Pro Ser His Arg Pro Leu Phe Phe Ile Leu Val Tyr Leu 1245 1250 1255 1260 ATT TCC ACT GCA GTG GTA ACA AGA TGG AAT CTG GAC ATA GCC GGA TTG 4201 Ile Ser Thr Ala Val Val Thr Arg Trp Asn Leu Asp Ile Ala Gly Leu 1265 1270 1275 CTG TTG CAG TGT GTC CCA ACC CTT TTG ATG GTT TTT ACG ATG TGG GCA 4249 Leu Leu Gln Cys Val Pro Thr Leu Leu Met Val Phe Thr Met Trp Ala 1280 1285 1290 GAC ATT CTC ACC CTG ATC CTC ATA CTG CCC ACT TAC GAG TTA ACG AAG 4297 Asp Ile Leu Thr Leu Ile Leu Ile Leu Pro Thr Tyr Glu Leu Thr Lys 1295 1300 1305 CTA TAT TAT CTT AAG GAA GTG AGG ATT GGG GCA GAA AAG GGC TGG TTA 4345 Leu Tyr Tyr Leu Lys Glu Val Arg Ile Gly Ala Glu Lys Gly Trp Leu 1310 1315 1320 TGG AAA ACC AAC TTC AAG AGG GTA AAC GAC ATA TAC GAA GTT GAC CAA 4393 Trp Lys Thr Asn Phe Lys Arg Val Asn Asp Ile Tyr Glu Val Asp Gln 1325 1330 1335 1340 GCT GGT GAA GGG GTA TAC CTA TTC CCG TCA AAA CAA AAG ACA AGT TCA 4441 Ala Gly Glu Gly Val Tyr Leu Phe Pro Ser Lys Gln Lys Thr Ser Ser 1345 1350 1355 ATG ACA GGC ACC ATG TTG CCA TTG ATC AAA GCC ATA CTT ATC AGC TGC 4489 Met Thr Gly Thr Met Leu Pro Leu Ile Lys Ala Ile Leu Ile Ser Cys 1360 1365 1370 GTC AGT AAT AAG TGG CAG TTC ATA TAT CTA CTG TAC TTG ATA TTT GAA 4537 Val Ser Asn Lys Trp Gln Phe Ile Tyr Leu Leu Tyr Leu Ile Phe Glu 1375 1380 1385 GTA TCT TAC TAC CTC CAC AAG AAG ATC ATA GAT GAA ATA GCA GGA GGG 4585 Val Ser Tyr Tyr Leu His Lys Lys Ile Ile Asp Glu Ile Ala Gly Gly 1390 1395 1400 ACC AAC TTC ATC TCA AGA CTT GTA GCC GCT TTG ATC GAA GTC AAT TGG 4633 Thr Asn Phe Ile Ser Arg Leu Val Ala Ala Leu Ile Glu Val Asn Trp 1405 1410 1415 1420 GCC TTT GAC AAC GAA GAA GTT AGG GGT TTG AAG AAG TTC TTC CTG TTG 4681 Ala Phe Asp Asn Glu Glu Val Arg Gly Leu Lys Lys Phe Phe Leu Leu 1425 1430 1435 TCT AGT AGG GTT AAA GAA CTG ATC ATC AAA CAC AAA GTG AGG AAT GAA 4729 Ser Ser Arg Val Lys Glu Leu Ile Ile Lys His Lys Val Arg Asn Glu 1440 1445 1450 GTA ATG GTC CGC TGG TTT GGT GAC GAA GAG GTC TAT GGG ATG CCG AAG 4777 Val Met Val Arg Trp Phe Gly Asp Glu Glu Val Tyr Gly Met Pro Lys 1455 1460 1465 TTG GTT GGC CTA GTC AAG GCA GCA ACA TTG AGT AAA AAT AAA CAT TGT 4825 Leu Val Gly Leu Val Lys Ala Ala Thr Leu Ser Lys Asn Lys His Cys 1470 1475 1480 ATT TTG TGC ACC GTC TGT GAA GAC AGA GAG TGG AGA GGA GAA ACC TGC 4873 Ile Leu Cys Thr Val Cys Glu Asp Arg Glu Trp Arg Gly Glu Thr Cys 1485 1490 1495 1500 CCA AAA TGC GGG CGT TTT GGG CCA CCA ATG ACC TGT GGC ATG ACC CTA 4921 Pro Lys Cys Gly Arg Phe Gly Pro Pro Met Thr Cys Gly Met Thr Leu 1505 1510 1515 GCC GAC TTT GAA GAG AAA CAC TAT AAG AGG ATC TTT TTT AGA GAG GAT 4969 Ala Asp Phe Glu Glu Lys His Tyr Lys Arg Ile Phe Phe Arg Glu Asp 1520 1525 1530 CAA TCA GAA GGG CCG GTT AGG GAG GAG TAC GCA GGG TAT CTG CAA TAT 5017 Gln Ser Glu Gly Pro Val Arg Glu Glu Tyr Ala Gly Tyr Leu Gln Tyr 1535 1540 1545 AGA GCC AGA GGG CAA TTG TTC CTG AGG AAT CTC CCA GTG CTA GCA ACA 5065 Arg Ala Arg Gly Gln Leu Phe Leu Arg Asn Leu Pro Val Leu Ala Thr 1550 1555 1560 AAA GTC AAG ATG CTC CTG GTC GGC AAT CTT GGG ACG GAG GTG GGA GAT 5113 Lys Val Lys Met Leu Leu Val Gly Asn Leu Gly Thr Glu Val Gly Asp 1565 1570 1575 1580 TTG GAA CAC CTT GGC TGG GTG CTT AGA GGG CCT GCC GTT TGC AAG AAG 5161 Leu Glu His Leu Gly Trp Val Leu Arg Gly Pro Ala Val Cys Lys Lys 1585 1590 1595 GTC ACT GAA CAT GAG AAA TGT ACC ACA TCC ATG ATG GAC AAA TTG ACT 5209 Val Thr Glu His Glu Lys Cys Thr Thr Ser Met Met Asp Lys Leu Thr 1600 1605 1610 GCT TTT TTC GGT GTT ATG CCG AGG GGC ACC ACA CCT AGA GCC CCT GTG 5257 Ala Phe Phe Gly Val Met Pro Arg Gly Thr Thr Pro Arg Ala Pro Val 1615 1620 1625 AGA TTC CCT ACC TCT CTC TTA AAG ATA AGA AGG GGT TTG GAA ACT GGC 5305 Arg Phe Pro Thr Ser Leu Leu Lys Ile Arg Arg Gly Leu Glu Thr Gly 1630 1635 1640 TGG GCG TAC ACA CAC CAA GGT GGC ATC AGT TCA GTG GAC CAT GTC ACT 5353 Trp Ala Tyr Thr His Gln Gly Gly Ile Ser Ser Val Asp His Val Thr 1645 1650 1655 1660 TGT GGA AAA GAC TTA CTG GTA TGT GAC ACT ATG GGC CGG ACA AGG GTT 5401 Cys Gly Lys Asp Leu Leu Val Cys Asp Thr Met Gly Arg Thr Arg Val 1665 1670 1675 GTT TGC CAG TCA AAT AAT AAG ATG ACA GAT GAG TCC GAG TAT GGA GTT 5449 Val Cys Gln Ser Asn Asn Lys Met Thr Asp Glu Ser Glu Tyr Gly Val 1680 1685 1690 AAA ACT GAC TCC GGA TGC CCG GAA GGA GCT AGG TGT TAT GTG TTT AAC 5497 Lys Thr Asp Ser Gly Cys Pro Glu Gly Ala Arg Cys Tyr Val Phe Asn 1695 1700 1705 CCA GAG GCG GTT AAC ATA TCA GGG ACT AAA GGA GCC ATG GTC CAC TTA 5545 Pro Glu Ala Val Asn Ile Ser Gly Thr Lys Gly Ala Met Val His Leu 1710 1715 1720 CAA AAA ACT GGA GGA GAA TTC ACC TGT GTG ACA GCA TCA GGA ACT CCG 5593 Gln Lys Thr Gly Gly Glu Phe Thr Cys Val Thr Ala Ser Gly Thr Pro 1725 1730 1735 1740 GCT TTC TTT GAT CTT AAA AAC CTT AAA GGC TGG TCA GGG CTA CCG ATA 5641 Ala Phe Phe Asp Leu Lys Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile 1745 1750 1755 TTT GAG GCA TCA AGT GGA AGG GTA GTC GGC AGG GTC AAG GTC GGT AAG 5689 Phe Glu Ala Ser Ser Gly Arg Val Val Gly Arg Val Lys Val Gly Lys 1760 1765 1770 AAT GAG GAC TCT AAA CCA ACC AAG CTT ATG AGT GGA ATA CAA ACA GTT 5737 Asn Glu Asp Ser Lys Pro Thr Lys Leu Met Ser Gly Ile Gln Thr Val 1775 1780 1785 TCC AAA AGT ACC ACA GAC TTG ACA GAA ATG GTA AAG AAA ATA ACT ACC 5785 Ser Lys Ser Thr Thr Asp Leu Thr Glu Met Val Lys Lys Ile Thr Thr 1790 1795 1800 ATG AGC AGG GGA GAA TTC AGA CAA ATA ACC CTT GCT ACA GGT GCC GGA 5833 Met Ser Arg Gly Glu Phe Arg Gln Ile Thr Leu Ala Thr Gly Ala Gly 1805 1810 1815 1820 AAA ACC ACG GAA CTC CCT AGG TCA GTC ATA GAA GAG ATA GGG AGG CAT 5881 Lys Thr Thr Glu Leu Pro Arg Ser Val Ile Glu Glu Ile Gly Arg His 1825 1830 1835 AAG AGA GTC TTG GTC TTG ATT CCT CTG AGG GCG GCA GCA GAG TCA GTA 5929 Lys Arg Val Leu Val Leu Ile Pro Leu Arg Ala Ala Ala Glu Ser Val 1840 1845 1850 TAC CAA TAT ATG AGA CAA AAA CAT CCA AGC ATC GCA TTT AAC CTG AGG 5977 Tyr Gln Tyr Met Arg Gln Lys His Pro Ser Ile Ala Phe Asn Leu Arg 1855 1860 1865 ATA GGG GAG ATG AAG GAA GGG GAC ATG GCC ACA GGG ATA ACT TAT GCT 6025 Ile Gly Glu Met Lys Glu Gly Asp Met Ala Thr Gly Ile Thr Tyr Ala 1870 1875 1880 TCA TAC GGT TAC TTC TGT CAG ATG CCA CAA CCT AAG TTG CGA GCC GCA 6073 Ser Tyr Gly Tyr Phe Cys Gln Met Pro Gln Pro Lys Leu Arg Ala Ala 1885 1890 1895 1900 ATG GTT GAG TAC TCC TTC ATA TTT CTT GAT GAG TAC CAC TGT GCC ACC 6121 Met Val Glu Tyr Ser Phe Ile Phe Leu Asp Glu Tyr His Cys Ala Thr 1905 1910 1915 CCT GAA CAA TTG GCT ATC ATG GGA AAG ATT CAC AGA TTT TCA GAG AAC 6169 Pro Glu Gln Leu Ala Ile Met Gly Lys Ile His Arg Phe Ser Glu Asn 1920 1925 1930 CTG CGG GTG GTG GCC ATG ACC GCA ACA CCA GTA GGC ACG GTA ACG ACC 6217 Leu Arg Val Val Ala Met Thr Ala Thr Pro Val Gly Thr Val Thr Thr 1935 1940 1945 ACA GGG CAG AAA CAC CCT ATA GAA GAA TTC ATA GCC CCA GAT GTG ATG 6265 Thr Gly Gln Lys His Pro Ile Glu Glu Phe Ile Ala Pro Asp Val Met 1950 1955 1960 AAA GGG AAA GAC TTA GGT TCA GAG TAC TTG GAC ATT GCT GGA TTA AAG 6313 Lys Gly Lys Asp Leu Gly Ser Glu Tyr Leu Asp Ile Ala Gly Leu Lys 1965 1970 1975 1980 ATA CCA GTA GAG GAG ATG AAG AGC AAT ATG CTG GTT TTT GTG CCC ACC 6361 Ile Pro Val Glu Glu Met Lys Ser Asn Met Leu Val Phe Val Pro Thr 1985 1990 1995 AGG AAC ATG GCA GTG GAG ACA GCA AAG AAA TTG AAA GCT AAG GGT TAT 6409 Arg Asn Met Ala Val Glu Thr Ala Lys Lys Leu Lys Ala Lys Gly Tyr 2000 2005 2010 AAC TCA GGC TAC TAT TAT AGT GGT GAG GAT CCA TCT AAC CTG AGG GTG 6457 Asn Ser Gly Tyr Tyr Tyr Ser Gly Glu Asp Pro Ser Asn Leu Arg Val 2015 2020 2025 GTA ACA TCG CAG TCC CCG TAC GTG GTG GTG GCA ACC AAC GCG ATA GAA 6505 Val Thr Ser Gln Ser Pro Tyr Val Val Val Ala Thr Asn Ala Ile Glu 2030 2035 2040 TCA GGT GTT ACT CTC CCG GAC TTG GAT GTG GTT GTC GAT ACA GGG CTT 6553 Ser Gly Val Thr Leu Pro Asp Leu Asp Val Val Val Asp Thr Gly Leu 2045 2050 2055 2060 AAG TGT GAA AAG AGA ATA CGG CTG TCA CCT AAG ATG CCC TTC ATA GTG 6601 Lys Cys Glu Lys Arg Ile Arg Leu Ser Pro Lys Met Pro Phe Ile Val 2065 2070 2075 ACG GGC CTG AAG AGA ATG GCT GTC ACG ATT GGG GAA CAA GCC CAG AGA 6649 Thr Gly Leu Lys Arg Met Ala Val Thr Ile Gly Glu Gln Ala Gln Arg 2080 2085 2090 AGG GGG AGA GTT GGG AGA GTA AAG CCT GGA AGA TAC TAC AGG AGT CAA 6697 Arg Gly Arg Val Gly Arg Val Lys Pro Gly Arg Tyr Tyr Arg Ser Gln 2095 2100 2105 GAA ACT CCC GTT GGT TCT AAA GAT TAC CAT TAT GAT TTA CTG CAA GCA 6745 Glu Thr Pro Val Gly Ser Lys Asp Tyr His Tyr Asp Leu Leu Gln Ala 2110 2115 2120 CAG AGG TAC GGT ATT GAA GAT GGG ATA AAC ATC ACC AAA TCC TTT AGA 6793 Gln Arg Tyr Gly Ile Glu Asp Gly Ile Asn Ile Thr Lys Ser Phe Arg 2125 2130 2135 2140 GAG ATG AAC TAT GAT TGG AGC CTT TAT GAG GAG GAC AGT CTG ATG ATT 6841 Glu Met Asn Tyr Asp Trp Ser Leu Tyr Glu Glu Asp Ser Leu Met Ile 2145 2150 2155 ACA CAA TTG GAA ATT CTT AAT AAT TTG TTG ATA TCA GAT GAA CTA CCA 6889 Thr Gln Leu Glu Ile Leu Asn Asn Leu Leu Ile Ser Asp Glu Leu Pro 2160 2165 2170 ATG GCA GTA AAA AAT ATA ATG GCC AGG ACT GAC CAC CCA GAA CCA ATT 6937 Met Ala Val Lys Asn Ile Met Ala Arg Thr Asp His Pro Glu Pro Ile 2175 2180 2185 CAG CTG GCG TAC AAC AGC TAC GAA ACA CAA GTG CCA GTG CTA TTC CCA 6985 Gln Leu Ala Tyr Asn Ser Tyr Glu Thr Gln Val Pro Val Leu Phe Pro 2190 2195 2200 AAA ATA AAG AAT GGA GAG GTG ACT GAC AGT TAC GAT AAC TAT ACC TTC 7033 Lys Ile Lys Asn Gly Glu Val Thr Asp Ser Tyr Asp Asn Tyr Thr Phe 2205 2210 2215 2220 CTC AAC GCA AGA AAA TTA GGG GAT GAT GTA CCC CCT TAC GTG TAT GCC 7081 Leu Asn Ala Arg Lys Leu Gly Asp Asp Val Pro Pro Tyr Val Tyr Ala 2225 2230 2235 ACA GAG GAT GAG GAC TTA GCG GTA GAG CTG CTG GGC TTA GAC TGG CCG 7129 Thr Glu Asp Glu Asp Leu Ala Val Glu Leu Leu Gly Leu Asp Trp Pro 2240 2245 2250 GAC CCT GGA AAC CAA GGG ACC GTA GAG ACT GGC AGA GCA CTA AAA CAG 7177 Asp Pro Gly Asn Gln Gly Thr Val Glu Thr Gly Arg Ala Leu Lys Gln 2255 2260 2265 GTA GTT GGT CTA TCA ACA GCT GAG AAT GCC CTG TTA GTA GCC TTA TTC 7225 Val Val Gly Leu Ser Thr Ala Glu Asn Ala Leu Leu Val Ala Leu Phe 2270 2275 2280 GGC TAC GTA GGA TAT CAG GCG CTT TCA AAG AGG CAT ATA CCA GTA GTC 7273 Gly Tyr Val Gly Tyr Gln Ala Leu Ser Lys Arg His Ile Pro Val Val 2285 2290 2295 2300 ACA GAC ATA TAT TCA ATT GAA GAT CAC AGG TTG GAA GAC ACC ACA CAC 7321 Thr Asp Ile Tyr Ser Ile Glu Asp His Arg Leu Glu Asp Thr Thr His 2305 2310 2315 CTA CAG TAC GCC CCA AAT GCT ATC AAG ACG GAG GGG AAG GAG ACA GAA 7369 Leu Gln Tyr Ala Pro Asn Ala Ile Lys Thr Glu Gly Lys Glu Thr Glu 2320 2325 2330 TTG AAA GAG CTA GCT CAG GGG GAT GTG CAG AGA TGT GTG GAA GCC ATG 7417 Leu Lys Glu Leu Ala Gln Gly Asp Val Gln Arg Cys Val Glu Ala Met 2335 2340 2345 ACC AAT TAT GCA AGA GAG GGT ATC CAA TTT ATG AAG TCT CAG GCA CTG 7465 Thr Asn Tyr Ala Arg Glu Gly Ile Gln Phe Met Lys Ser Gln Ala Leu 2350 2355 2360 AAG GTG AAG GAA ACC CCT ACT TAC AAG GAG ACA ATG GAC ACT GTG ACG 7513 Lys Val Lys Glu Thr Pro Thr Tyr Lys Glu Thr Met Asp Thr Val Thr 2365 2370 2375 2380 GAC TAT GTA AAG AAA TTC ATG GAG GCG CTG GCA GAC AGT AAA GAA GAC 7561 Asp Tyr Val Lys Lys Phe Met Glu Ala Leu Ala Asp Ser Lys Glu Asp 2385 2390 2395 ATC ATA AAA TAT GGG CTG TGG GGG ACG CAC ACA GCC TTA TAT AAG AGC 7609 Ile Ile Lys Tyr Gly Leu Trp Gly Thr His Thr Ala Leu Tyr Lys Ser 2400 2405 2410 ATC AGT GCC AGG CTT GGG GGT GAG ACT GCG TTC GCT ACC CTG GTA GTG 7657 Ile Ser Ala Arg Leu Gly Gly Glu Thr Ala Phe Ala Thr Leu Val Val 2415 2420 2425 AAG TGG CTG GCA TTT GGG GGT GAA TCA ATA GCA GAC CAT GTC AAA CAA 7705 Lys Trp Leu Ala Phe Gly Gly Glu Ser Ile Ala Asp His Val Lys Gln 2430 2435 2440 GCG GCC ACA GAC TTG GTC GTT TAC TAT ATC ATC AAC AGA CCT CAG TTC 7753 Ala Ala Thr Asp Leu Val Val Tyr Tyr Ile Ile Asn Arg Pro Gln Phe 2445 2450 2455 2460 CCA GGA GAC ACG GAG ACA CAA CAA GAC GGA AGG AAA TTT GTG GCC AGC 7801 Pro Gly Asp Thr Glu Thr Gln Gln Asp Gly Arg Lys Phe Val Ala Ser 2465 2470 2475 CTA CTG GCC TCA GCT CTA GCT ACT TAC ACA TAC AAA AGC TGG AAT TAC 7849 Leu Leu Ala Ser Ala Leu Ala Thr Tyr Thr Tyr Lys Ser Trp Asn Tyr 2480 2485 2490 AAT AAC CTG TCC AAG ATA GTT GAA CCG GCT TTG GCC ACT CTG CCC TAT 7897 Asn Asn Leu Ser Lys Ile Val Glu Pro Ala Leu Ala Thr Leu Pro Tyr 2495 2500 2505 GCC GCC ACA GCT CTC AAA TTA TTC GCC CCC ACC CGA TTG GAG AGC GTT 7945 Ala Ala Thr Ala Leu Lys Leu Phe Ala Pro Thr Arg Leu Glu Ser Val 2510 2515 2520 GTC ATA TTA AGT ACC GCA ATC TAC AAA ACC TAC CTA TCA ATC AGG CGC 7993 Val Ile Leu Ser Thr Ala Ile Tyr Lys Thr Tyr Leu Ser Ile Arg Arg 2525 2530 2535 2540 GGA AAA AGC GAT GGT TTG CTA GGC ACG GGG GTT AGT GCG GCT ATG GAG 8041 Gly Lys Ser Asp Gly Leu Leu Gly Thr Gly Val Ser Ala Ala Met Glu 2545 2550 2555 ATC ATG TCA CAA AAT CCA GTA TCC GTG GGC ATA GCA GTC ATG CTA GGG 8089 Ile Met Ser Gln Asn Pro Val Ser Val Gly Ile Ala Val Met Leu Gly 2560 2565 2570 GTA GGG GCC GTG GCA GCC CAC AAT GCA ATC GAA GCC AGT GAG CAG AAA 8137 Val Gly Ala Val Ala Ala His Asn Ala Ile Glu Ala Ser Glu Gln Lys 2575 2580 2585 AGA ACA CTA CTC ATG AAA GTC TTT GTA AAG AAC TTC TTG GAC CAG GCA 8185 Arg Thr Leu Leu Met Lys Val Phe Val Lys Asn Phe Leu Asp Gln Ala 2590 2595 2600 GCC ACA GAT GAA TTA GTC AAG GAG AGT CCT GAA AAA ATA ATA ATG GCT 8233 Ala Thr Asp Glu Leu Val Lys Glu Ser Pro Glu Lys Ile Ile Met Ala 2605 2610 2615 2620 TTG TTT GAA GCA GTG CAG ACA GTC GGC AAC CCT CTT AGA CTT GTA TAC 8281 Leu Phe Glu Ala Val Gln Thr Val Gly Asn Pro Leu Arg Leu Val Tyr 2625 2630 2635 CAC CTT TAT GGA GTT TTT TAT AAA GGG TGG GAG GCA AAA GAG TTG GCC 8329 His Leu Tyr Gly Val Phe Tyr Lys Gly Trp Glu Ala Lys Glu Leu Ala 2640 2645 2650 CAA AGG ACA GCC GGT AGG AAC CTT TTC ACT TTA ATC ATG TTC GAG GCT 8377 Gln Arg Thr Ala Gly Arg Asn Leu Phe Thr Leu Ile Met Phe Glu Ala 2655 2660 2665 GTG GAA CTG CTG GGA GTA GAC AGT GAA GGA AAG GTC CGC CAG CTA TCA 8425 Val Glu Leu Leu Gly Val Asp Ser Glu Gly Lys Val Arg Gln Leu Ser 2670 2675 2680 AGT AAT TAC ATA CTA GAG CTT TTG TAT AAG TTC CGT GAC AGT ATC AAG 8473 Ser Asn Tyr Ile Leu Glu Leu Leu Tyr Lys Phe Arg Asp Ser Ile Lys 2685 2690 2695 2700 TCT AGC GTG AGG GAG ATG GCA ATC AGC TGG GCC CCT GCC CCT TTC AGT 8521 Ser Ser Val Arg Glu Met Ala Ile Ser Trp Ala Pro Ala Pro Phe Ser 2705 2710 2715 TGT GAT TGG ACA CCG ACG GAT GAC AGA ATA GGG CTC CCC CAA GAC AAT 8569 Cys Asp Trp Thr Pro Thr Asp Asp Arg Ile Gly Leu Pro Gln Asp Asn 2720 2725 2730 TTC CAC CAA GTG GAG ACG AAA TGC CCC TGT GGT TAC AAG ATG AAG GCA 8617 Phe His Gln Val Glu Thr Lys Cys Pro Cys Gly Tyr Lys Met Lys Ala 2735 2740 2745 GTT AAG AAT TGT GCT GGA GAA CTG AGA CTC TTG GAG GAG GAG GGT TCA 8665 Val Lys Asn Cys Ala Gly Glu Leu Arg Leu Leu Glu Glu Glu Gly Ser 2750 2755 2760 TTT CTC TGC AGA AAT AAA TTC GGG AGA GGT TCA CGG AAC TAC AGA GTG 8713 Phe Leu Cys Arg Asn Lys Phe Gly Arg Gly Ser Arg Asn Tyr Arg Val 2765 2770 2775 2780 ACA AAA TAT TAT GAT GAC AAC CTA TTA GAA ATA AAG CCA GTG ATA AGA 8761 Thr Lys Tyr Tyr Asp Asp Asn Leu Leu Glu Ile Lys Pro Val Ile Arg 2785 2790 2795 ATG GAA GGG CAT GTG GAA CTC TAC TAC AAG GGG GCC ACC ATC AAA CTG 8809 Met Glu Gly His Val Glu Leu Tyr Tyr Lys Gly Ala Thr Ile Lys Leu 2800 2805 2810 GAT TTC AAC AAC AGC AAA ACA ATA TTG GCA ACC GAT AAA TGG GAG GTT 8857 Asp Phe Asn Asn Ser Lys Thr Ile Leu Ala Thr Asp Lys Trp Glu Val 2815 2820 2825 GAT CAC TCC ACT CTG GTC AGG GTG CTC AAG AGG CAC ACA GGG GCT GGA 8905 Asp His Ser Thr Leu Val Arg Val Leu Lys Arg His Thr Gly Ala Gly 2830 2835 2840 TAT CAT GGG GCA TAC CTG GGC GAG AAA CCG AAC CAC AAA CAC CTG ATA 8953 Tyr His Gly Ala Tyr Leu Gly Glu Lys Pro Asn His Lys His Leu Ile 2845 2850 2855 2860 GAG AGG GAC TGT GCA ACC ATC ACC AAA GAT AAG GTC TGT TTT CTC AAA 9001 Glu Arg Asp Cys Ala Thr Ile Thr Lys Asp Lys Val Cys Phe Leu Lys 2865 2870 2875 ATG AAG AGA GGG TGC GCA TTT ACT TAT GAC TTA TCC CTT CAC AAC CTT 9049 Met Lys Arg Gly Cys Ala Phe Thr Tyr Asp Leu Ser Leu His Asn Leu 2880 2885 2890 ACC CGA CTG ATT GAA TTG GTA CAC AAG AAT AAC TTG GAA GAC AAA GAG 9097 Thr Arg Leu Ile Glu Leu Val His Lys Asn Asn Leu Glu Asp Lys Glu 2895 2900 2905 ATT CCC GCT GCT ACG GTT ACA ACC TGG CTG GCT TAC ACA TTT GTA AAT 9145 Ile Pro Ala Ala Thr Val Thr Thr Trp Leu Ala Tyr Thr Phe Val Asn 2910 2915 2920 GAA GAT ATA GGG ACC ATA AAA CCA GCC TTC GGG GAG AAA GTA ACG CTG 9193 Glu Asp Ile Gly Thr Ile Lys Pro Ala Phe Gly Glu Lys Val Thr Leu 2925 2930 2935 2940 GAG ATG CAG GAG GAG ATA ACC TTG CAG CCT GCT GTT GTG GTG GAT ACA 9241 Glu Met Gln Glu Glu Ile Thr Leu Gln Pro Ala Val Val Val Asp Thr 2945 2950 2955 ACA GAC GTA GCC GTG ACT GTG GTA GGG GAA GCC CCC ACT ATG ACT ACA 9289 Thr Asp Val Ala Val Thr Val Val Gly Glu Ala Pro Thr Met Thr Thr 2960 2965 2970 GGG GAG ACA CCG ACA GTG TTC ACC AGC TCA GGT TCA GGC CTG AAA AGC 9337 Gly Glu Thr Pro Thr Val Phe Thr Ser Ser Gly Ser Gly Leu Lys Ser 2975 2980 2985 CAA CAA GTT TTG AAA CTA GGG GTA GGT GAA GGC CAA TAT CCA GGG ACT 9385 Gln Gln Val Leu Lys Leu Gly Val Gly Glu Gly Gln Tyr Pro Gly Thr 2990 2995 3000 AAT CCA CAG AGG GCA AGC CTG CAC GAA GCC ATA CAA GGT GCA GAT GAG 9433 Asn Pro Gln Arg Ala Ser Leu His Glu Ala Ile Gln Gly Ala Asp Glu 3005 3010 3015 3020 AGG CCC TCG GTG CTG ATA TTG GGG TCT GAT AAA GCC ACC TCT AAT AGA 9481 Arg Pro Ser Val Leu Ile Leu Gly Ser Asp Lys Ala Thr Ser Asn Arg 3025 3030 3035 GTG AAG ACT GCA AAG AAT GTA AAG GTA TAC AGA GGC AGG GAC CCA CTA 9529 Val Lys Thr Ala Lys Asn Val Lys Val Tyr Arg Gly Arg Asp Pro Leu 3040 3045 3050 GAA GTG AGA GAT ATG ATG AGG AGG GGA AAG ATC CTG GTC GTA GCC CTG 9577 Glu Val Arg Asp Met Met Arg Arg Gly Lys Ile Leu Val Val Ala Leu 3055 3060 3065 TCT AGG GTT GAT AAT GCT CTA TTG AAA TTT GTT GAC TAC AAA GGC ACC 9625 Ser Arg Val Asp Asn Ala Leu Leu Lys Phe Val Asp Tyr Lys Gly Thr 3070 3075 3080 TTT CTA ACT AGG GAG GCC CTA GAG GCA TTA AGT TTG GGC AGG CCT AAA 9673 Phe Leu Thr Arg Glu Ala Leu Glu Ala Leu Ser Leu Gly Arg Pro Lys 3085 3090 3095 3100 AAG AAA AAC ATA ACC AAG GCA GAA GCG CAG TGG TTG CTG TGC CCC GAG 9721 Lys Lys Asn Ile Thr Lys Ala Glu Ala Gln Trp Leu Leu Cys Pro Glu 3105 3110 3115 GAC CAA ATG GAA GAG CTA CCC GAC TGG TTC GCA GCC GGG GAA CCC ATT 9769 Asp Gln Met Glu Glu Leu Pro Asp Trp Phe Ala Ala Gly Glu Pro Ile 3120 3125 3130 TTT TTA GAG GCC AAC ATT AAA CAT GAC AGG TAC CAT CTG GTG GGG GAT 9817 Phe Leu Glu Ala Asn Ile Lys His Asp Arg Tyr His Leu Val Gly Asp 3135 3140 3145 ATA GCT ACC ATC AAG GAA AAA GCC AAA CAG TTG GGG GCT ACA GAC TCC 9865 Ile Ala Thr Ile Lys Glu Lys Ala Lys Gln Leu Gly Ala Thr Asp Ser 3150 3155 3160 ACA AAG ATA TCT AAG GAG GTT GGT GCT AAA GTG TAT TCT ATG AAA CTG 9913 Thr Lys Ile Ser Lys Glu Val Gly Ala Lys Val Tyr Ser Met Lys Leu 3165 3170 3175 3180 AGT AAT TGG GTG ATG CAA GAA GAA AAT AAA CAG GGC AAT CTG ACC CCC 9961 Ser Asn Trp Val Met Gln Glu Glu Asn Lys Gln Gly Asn Leu Thr Pro 3185 3190 3195 TTG TTT GAA GAG CTC CTG CAA CAG TGT CCA CCC GGG GGC CAG AAC AAA 10009 Leu Phe Glu Glu Leu Leu Gln Gln Cys Pro Pro Gly Gly Gln Asn Lys 3200 3205 3210 ACT GCA CAC ATG GTC TCT GCT TAC CAA CTA GCT CAA GGG AAC TGG ATG 10057 Thr Ala His Met Val Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Met 3215 3220 3225 CCG ACC AGC TGC CAT GTT TTC ATG GGG ACC GTA TCT GCC AGG AGA ACC 10105 Pro Thr Ser Cys His Val Phe Met Gly Thr Val Ser Ala Arg Arg Thr 3230 3235 3240 AAG ACC CAC CCA TAC GAA GCA TAC GTT AAG TTA AGG GAG TTG GTA GAG 10153 Lys Thr His Pro Tyr Glu Ala Tyr Val Lys Leu Arg Glu Leu Val Glu 3245 3250 3255 3260 GAG CAC AAG ATG AAA ACA CTG TGT CCC GGA TCA AGC CTG GGT AGG CAC 10201 Glu His Lys Met Lys Thr Leu Cys Pro Gly Ser Ser Leu Gly Arg His 3265 3270 3275 AAC GAT TGG ATA ATT GGA AAA ATT AAA TAC CAG GGA AAC CTG AGG ACC 10249 Asn Asp Trp Ile Ile Gly Lys Ile Lys Tyr Gln Gly Asn Leu Arg Thr 3280 3285 3290 AAA CAC ATG TTG AAC CCC GGC AAG GTG GCA GAG CAA CTG TGC AGA GAG 10297 Lys His Met Leu Asn Pro Gly Lys Val Ala Glu Gln Leu Cys Arg Glu 3295 3300 3305 GGA CAC AGA CAC AAT GTG TAT AAC AAG ACA ATA AGC TCA GTA ATG ACA 10345 Gly His Arg His Asn Val Tyr Asn Lys Thr Ile Ser Ser Val Met Thr 3310 3315 3320 GCT ACT GGT ATC AGG TTG GAG AAG TTG CCC GTG GTT AGG GCC CAG ACA 10393 Ala Thr Gly Ile Arg Leu Glu Lys Leu Pro Val Val Arg Ala Gln Thr 3325 3330 3335 3340 GAC CCA ACC AAC TTC CAC CAA GCA ATA AGG GAT AAG ATA GAC AAG GAA 10441 Asp Pro Thr Asn Phe His Gln Ala Ile Arg Asp Lys Ile Asp Lys Glu 3345 3350 3355 GAG AAC CTA CAA ACC CCG GGT TTA CAT AAG AAA TTA ATG GAA GTT TTC 10489 Glu Asn Leu Gln Thr Pro Gly Leu His Lys Lys Leu Met Glu Val Phe 3360 3365 3370 AAC GCA TTG AAA CGA CCC GAG TTA GAG TCC TCC TAC GAC GCC GTG GAA 10537 Asn Ala Leu Lys Arg Pro Glu Leu Glu Ser Ser Tyr Asp Ala Val Glu 3375 3380 3385 TGG GAG GAA CTG GAG AGA GGA ATA AAC AGG AAG GGT GCT GCT GGT TTT 10585 Trp Glu Glu Leu Glu Arg Gly Ile Asn Arg Lys Gly Ala Ala Gly Phe 3390 3395 3400 TTC GAA CGC AAA AAT ATA GGG GAA ATA TTG GAT TCA GAG AAA AAT AAA 10633 Phe Glu Arg Lys Asn Ile Gly Glu Ile Leu Asp Ser Glu Lys Asn Lys 3405 3410 3415 3420 GTC GAA GAG ATT ATT GAC AAT CTG AAA AAA GGT AGA AAC ATT AAA TAT 10681 Val Glu Glu Ile Ile Asp Asn Leu Lys Lys Gly Arg Asn Ile Lys Tyr 3425 3430 3435 TAT GAA ACC GCG ATC CCA AAG AAT GAG AAG AGG GAC GTC AAC GAT GAC 10729 Tyr Glu Thr Ala Ile Pro Lys Asn Glu Lys Arg Asp Val Asn Asp Asp 3440 3445 3450 TGG ACC GCC GGT GAT TTC GTG GAC GAG AAG AAA CCT AGA GTC ATA CAA 10777 Trp Thr Ala Gly Asp Phe Val Asp Glu Lys Lys Pro Arg Val Ile Gln 3455 3460 3465 TAC CCT GAA GCA AAA ACA AGA CTG GCC ATC ACC AAG GTG ATG TAT AAG 10825 Tyr Pro Glu Ala Lys Thr Arg Leu Ala Ile Thr Lys Val Met Tyr Lys 3470 3475 3480 TGG GTG AAG CAG AAG CCA GTA GTT ATA CCC GGG TAT GAA GGG AAG ACA 10873 Trp Val Lys Gln Lys Pro Val Val Ile Pro Gly Tyr Glu Gly Lys Thr 3485 3490 3495 3500 CCT CTA TTC CAA ATT TTT GAC AAA GTA AAG AAG GAA TGG GAT CAA TTT 10921 Pro Leu Phe Gln Ile Phe Asp Lys Val Lys Lys Glu Trp Asp Gln Phe 3505 3510 3515 CAA AAT CCA GTG GCA GTG AGC TTC GAC ACT AAG GCG TGG GAC ACC CAG 10969 Gln Asn Pro Val Ala Val Ser Phe Asp Thr Lys Ala Trp Asp Thr Gln 3520 3525 3530 GTA ACC ACA AAA GAT TTG GAG CTG ATA AGG GAC ATA CAA AAG TAT TAT 11017 Val Thr Thr Lys Asp Leu Glu Leu Ile Arg Asp Ile Gln Lys Tyr Tyr 3535 3540 3545 TTC AAG AAG AAA TGG CAC AAA TTT ATT GAC ACC CTG ACC ACG CAT ATG 11065 Phe Lys Lys Lys Trp His Lys Phe Ile Asp Thr Leu Thr Thr His Met 3550 3555 3560 TCA GAA GTA CCC GTG ATC AGT GCT GAT GGG GAA GTA TAC ATA AGG AAA 11113 Ser Glu Val Pro Val Ile Ser Ala Asp Gly Glu Val Tyr Ile Arg Lys 3565 3570 3575 3580 GGG CAA AGA GGC AGT GGA CAA CCT GAC ACA AGT GCG GGC AAC AGC ATG 11161 Gly Gln Arg Gly Ser Gly Gln Pro Asp Thr Ser Ala Gly Asn Ser Met 3585 3590 3595 CTA AAT GTC TTA ACA ATG GTT TAC GCC TTC TGC GAG GCC ACA GGA GTA 11209 Leu Asn Val Leu Thr Met Val Tyr Ala Phe Cys Glu Ala Thr Gly Val 3600 3605 3610 CCC TAC AAG AGC TTT GAC AGG GTG GCA AAA ATT CAT GTG TGC GGG GAT 11257 Pro Tyr Lys Ser Phe Asp Arg Val Ala Lys Ile His Val Cys Gly Asp 3615 3620 3625 GAT GGC TTC CTG ATC ACA GAA AGA GCT CTC GGT GAG AAA TTT GCA AGT 11305 Asp Gly Phe Leu Ile Thr Glu Arg Ala Leu Gly Glu Lys Phe Ala Ser 3630 3635 3640 AAG GGA GTC CAG ATC CTT TAT GAA GCT GGG AAG CCC CAG AAG ATC ACT 11353 Lys Gly Val Gln Ile Leu Tyr Glu Ala Gly Lys Pro Gln Lys Ile Thr 3645 3650 3655 3660 GAA GGG GAC AAA ATG AAA GTG GCC TAC CAA TTT GAT GAT ATT GAG TTT 11401 Glu Gly Asp Lys Met Lys Val Ala Tyr Gln Phe Asp Asp Ile Glu Phe 3665 3670 3675 TGC TCC CAT ACA CCA ATA CAA GTA AGA TGG TCA GAT AAC ACT TCT AGT 11449 Cys Ser His Thr Pro Ile Gln Val Arg Trp Ser Asp Asn Thr Ser Ser 3680 3685 3690 TAC ATG CCG GGG AGA AAT ACA ACC ACA ATC CTG GCA AAG ATG GCC ACG 11497 Tyr Met Pro Gly Arg Asn Thr Thr Thr Ile Leu Ala Lys Met Ala Thr 3695 3700 3705 AGG TTA GAT TCC AGC GGT GAA AGG GGT ACC ATA GCA TAT GAG AAA GCA 11545 Arg Leu Asp Ser Ser Gly Glu Arg Gly Thr Ile Ala Tyr Glu Lys Ala 3710 3715 3720 GTA GCA TTT AGC TTC CTG CTG ATG TAC TCC TGG AAC CCA CTA ATT AGA 11593 Val Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp Asn Pro Leu Ile Arg 3725 3730 3735 3740 AGG ATC TGC TTA CTG GTG CTA TCA ACT GAA CTG CAA GTG AAA CCA GGG 11641 Arg Ile Cys Leu Leu Val Leu Ser Thr Glu Leu Gln Val Lys Pro Gly 3745 3750 3755 AAG TCA ACT ACT TAC TAT TAT GAA GGA GAC CCG ATA TCT GCC TAC AAG 11689 Lys Ser Thr Thr Tyr Tyr Tyr Glu Gly Asp Pro Ile Ser Ala Tyr Lys 3760 3765 3770 GAA GTC ATC GGC CAC AAC CTT TTT GAT CTT AAG AGA ACA AGC TTT GAG 11737 Glu Val Ile Gly His Asn Leu Phe Asp Leu Lys Arg Thr Ser Phe Glu 3775 3780 3785 AAG CTG GCC AAG TTA AAT CTT AGC ATG TCT GTA CTC GGG GCC TGG ACT 11785 Lys Leu Ala Lys Leu Asn Leu Ser Met Ser Val Leu Gly Ala Trp Thr 3790 3795 3800 AGA CAC ACC AGT AAA AGA CTA CTA CAA GAC TGT GTC AAT ATA GGT GTT 11833 Arg His Thr Ser Lys Arg Leu Leu Gln Asp Cys Val Asn Ile Gly Val 3805 3810 3815 3820 AAA GAG GGC AAT TGG CTA GTC AAT GCA GAC AGA CTA GTA AGT AGC AAG 11881 Lys Glu Gly Asn Trp Leu Val Asn Ala Asp Arg Leu Val Ser Ser Lys 3825 3830 3835 ACC GGG AAT AGG TAC ATA CCC GGA GAG GGT CAC ACC CTG CAA GGA AGA 11929 Thr Gly Asn Arg Tyr Ile Pro Gly Glu Gly His Thr Leu Gln Gly Arg 3840 3845 3850 CAT TAT GAA GAA CTG GTG TTG GCA AGA AAA CAG ATC AAC AAC TTT CAA 11977 His Tyr Glu Glu Leu Val Leu Ala Arg Lys Gln Ile Asn Asn Phe Gln 3855 3860 3865 GGG ACA GAC AGG TAC AAC CTA GGC CCA ATA GTC AAC ATG GTG TTA AGG 12025 Gly Thr Asp Arg Tyr Asn Leu Gly Pro Ile Val Asn Met Val Leu Arg 3870 3875 3880 AGG CTG AGA GTC ATG ATG ATG ACG CTG ATA GGG AGA GGG GCA 12067 Arg Leu Arg Val Met Met Met Thr Leu Ile Gly Arg Gly Ala 3885 3890 3895 TGAGCGCGGG TAACCCGGGA TCTGAACCCG CCAGTAGGAC CCTATTGTAG ATAACACTAA 12127 TTTTCTTTTT TTTCTTTTTT ATTTATTTAG ATATTATTAT TTATTTATTT ATTTATTTAT 12187 TGAATGAGTA AGAACTGGTA TAAACTACCT CAAGTTACCA CACTACACTC ATTTTTAACA 12247 GCACTTTAGC TGGAAGGAAA ATTCCTGACG TCCACAGTTG GGCTAAGGTA ATTTCTAACG 12307 GCCC 12311 3898 amino acids amino acid linear protein unknown 2 Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Asn Lys Gln Lys 1 5 10 15 Pro Met Gly Val Glu Glu Pro Val Tyr Asp Ala Thr Gly Arg Pro Leu 20 25 30 Phe Gly Asp Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro 35 40 45 His Asp Arg Gly Arg Gly Asn Ile Lys Thr Thr Leu Lys Asn Leu Pro 50 55 60 Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly 65 70 75 80 Ile Tyr Val Lys Pro Gly Pro Val Phe Tyr Gln Asp Tyr Met Gly Pro 85 90 95 Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Val Gln Phe Cys 100 105 110 Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu 115 120 125 Tyr His Thr Tyr Val Cys Ile Asp Gly Cys Ile Leu Leu Lys Leu Ala 130 135 140 Lys Arg Gly Glu Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asp 145 150 155 160 Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser 165 170 175 Lys Glu Lys Lys Pro Asp Arg Ile Asn Lys Gly Lys Leu Lys Ile Ala 180 185 190 Pro Lys Glu His Glu Lys Asp Ser Arg Thr Arg Pro Pro Asp Ala Thr 195 200 205 Ile Val Val Glu Gly Val Lys Tyr Gln Val Lys Lys Lys Gly Lys Val 210 215 220 Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro 225 230 235 240 Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val 245 250 255 Ile Ala Ile Met Leu Tyr Gln Pro Val Glu Ala Glu Asn Ile Thr Gln 260 265 270 Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln His Ala Met Tyr 275 280 285 Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Gly Glu Ile 290 295 300 Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Val Glu Leu Lys Glu 305 310 315 320 Ile Gln Gly Met Met Asp Ala Ser Glu Gly Thr Asn Tyr Thr Cys Cys 325 330 335 Lys Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp His 340 345 350 Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Ala Asp Leu 355 360 365 Ala Glu Gly Pro Pro Val Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp 370 375 380 Lys Asp Ala Asp Ile Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr 385 390 395 400 Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr 405 410 415 Val Ile Glu Ser Pro Cys Asn Phe Asn Val Ser Val Glu Asp Thr Leu 420 425 430 Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Ala Ala Leu Tyr 435 440 445 Leu Val Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala 450 455 460 Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Arg Thr Ala Gly Lys 465 470 475 480 Arg Leu Glu Gly Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu Ser 485 490 495 Pro Tyr Cys Asn Val Thr Ser Lys Ile Gly Tyr Ile Trp Tyr Thr Asn 500 505 510 Asn Cys Thr Pro Ala Cys Leu Pro Lys Asn Thr Lys Ile Ile Gly Pro 515 520 525 Gly Lys Phe Asp Thr Asn Ala Glu Asp Gly Lys Ile Leu His Glu Met 530 535 540 Gly Gly His Leu Ser Glu Phe Leu Leu Leu Ser Leu Val Val Leu Ser 545 550 555 560 Asp Phe Ala Pro Glu Thr Ala Ser Ala Leu Tyr Leu Ile Leu His Tyr 565 570 575 Val Ile Pro Gln Pro His Asp Glu Pro Glu Gly Cys Asp Thr Asn Gln 580 585 590 Leu Asn Leu Thr Val Glu Leu Arg Thr Glu Asp Val Ile Pro Ser Ser 595 600 605 Val Trp Asn Val Gly Lys Tyr Val Cys Val Arg Pro Asp Trp Trp Pro 610 615 620 Tyr Glu Thr Glu Val Ala Leu Leu Phe Glu Glu Val Gly Gln Val Val 625 630 635 640 Lys Leu Ala Leu Arg Ala Leu Arg Asp Leu Thr Arg Val Trp Asn Ser 645 650 655 Ala Ser Thr Ile Ala Phe Leu Ile Cys Leu Ile Lys Val Leu Arg Gly 660 665 670 Gln Ile Val Gln Gly Val Val Trp Leu Leu Leu Val Thr Gly Ala Gln 675 680 685 Gly Arg Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala Ile Ser Ser Thr 690 695 700 Asp Glu Ile Gly Leu Leu Gly Ala Gly Gly Leu Thr Thr Thr Trp Lys 705 710 715 720 Glu Tyr Asn His Asp Leu Gln Leu Asn Asp Gly Thr Val Lys Ala Ser 725 730 735 Cys Val Ala Gly Ser Phe Lys Val Thr Ala Leu Asn Val Val Ser Arg 740 745 750 Arg Tyr Leu Ala Ser Leu His Lys Lys Ala Leu Pro Thr Ser Val Thr 755 760 765 Phe Glu Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu Met Gly 770 775 780 Asp Asp Phe Arg Ser Gly Leu Cys Pro Phe Asp Thr Ser Pro Val Val 785 790 795 800 Lys Gly Lys Tyr Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe Tyr Leu 805 810 815 Val Cys Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val Ser 820 825 830 Pro Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe Arg Arg Asp Lys 835 840 845 Pro Phe Pro His Arg Met Asp Cys Val Thr Thr Thr Val Glu Asn Glu 850 855 860 Asp Leu Phe Tyr Cys Lys Leu Gly Gly Asn Trp Thr Cys Val Lys Gly 865 870 875 880 Glu Pro Val Val Tyr Thr Gly Gly Leu Val Lys Gln Cys Arg Trp Cys 885 890 895 Gly Phe Asp Phe Asp Gly Pro Asp Gly Leu Pro His Tyr Pro Ile Gly 900 905 910 Lys Cys Ile Leu Ala Asn Glu Thr Gly Tyr Arg Ile Val Asp Ser Thr 915 920 925 Asp Cys Asn Arg Asp Gly Val Val Ile Ser Thr Glu Gly Ser His Glu 930 935 940 Cys Leu Ile Gly Asn Thr Thr Val Lys Val His Ala Ser Asp Glu Arg 945 950 955 960 Leu Gly Pro Met Pro Cys Arg Pro Lys Glu Ile Val Ser Ser Ala Gly 965 970 975 Pro Val Lys Lys Thr Ser Cys Thr Phe Asn Tyr Thr Lys Thr Leu Lys 980 985 990 Asn Arg Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe Gln Gln Tyr Met Leu 995 1000 1005 Lys Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Asp Ala Thr Asp Arg His 1010 1015 1020 Ser Asp Tyr Phe Ala Glu Phe Val Val Leu Val Val Val Ala Leu Leu 1025 1030 1035 1040 Gly Gly Arg Tyr Val Leu Trp Leu Ile Val Thr Tyr Val Val Leu Thr 1045 1050 1055 Glu Gln Leu Ala Ala Gly Leu Pro Leu Gly Gln Gly Glu Val Val Leu 1060 1065 1070 Ile Gly Asn Leu Ile Thr His Thr Asp Ile Glu Val Val Val Tyr Phe 1075 1080 1085 Leu Leu Leu Tyr Leu Val Met Arg Asp Glu Pro Ile Lys Lys Trp Ile 1090 1095 1100 Leu Leu Leu Phe His Ala Met Thr Asn Asn Pro Val Lys Thr Ile Thr 1105 1110 1115 1120 Val Ala Leu Leu Met Val Ser Gly Val Ala Lys Gly Gly Lys Ile Asp 1125 1130 1135 Gly Gly Trp Gln Arg Leu Pro Gly Thr Ser Phe Asp Ile Gln Leu Ala 1140 1145 1150 Leu Thr Val Ile Val Val Ala Val Met Leu Leu Ala Lys Arg Asp Pro 1155 1160 1165 Thr Thr Val Pro Leu Val Ile Thr Val Ala Pro Leu Arg Thr Ala Lys 1170 1175 1180 Met Thr Asn Gly Leu Ser Thr Asp Ile Ala Ile Ala Thr Val Ser Ala 1185 1190 1195 1200 Ala Leu Leu Thr Trp Thr Tyr Ile Ser Asp Tyr Tyr Arg Tyr Lys Thr 1205 1210 1215 Trp Leu Gln Tyr Leu Ile Ser Thr Val Thr Gly Ile Phe Leu Ile Arg 1220 1225 1230 Val Leu Lys Gly Ile Gly Glu Leu Asp Leu His Thr Pro Thr Leu Pro 1235 1240 1245 Ser His Arg Pro Leu Phe Phe Ile Leu Val Tyr Leu Ile Ser Thr Ala 1250 1255 1260 Val Val Thr Arg Trp Asn Leu Asp Ile Ala Gly Leu Leu Leu Gln Cys 1265 1270 1275 1280 Val Pro Thr Leu Leu Met Val Phe Thr Met Trp Ala Asp Ile Leu Thr 1285 1290 1295 Leu Ile Leu Ile Leu Pro Thr Tyr Glu Leu Thr Lys Leu Tyr Tyr Leu 1300 1305 1310 Lys Glu Val Arg Ile Gly Ala Glu Lys Gly Trp Leu Trp Lys Thr Asn 1315 1320 1325 Phe Lys Arg Val Asn Asp Ile Tyr Glu Val Asp Gln Ala Gly Glu Gly 1330 1335 1340 Val Tyr Leu Phe Pro Ser Lys Gln Lys Thr Ser Ser Met Thr Gly Thr 1345 1350 1355 1360 Met Leu Pro Leu Ile Lys Ala Ile Leu Ile Ser Cys Val Ser Asn Lys 1365 1370 1375 Trp Gln Phe Ile Tyr Leu Leu Tyr Leu Ile Phe Glu Val Ser Tyr Tyr 1380 1385 1390 Leu His Lys Lys Ile Ile Asp Glu Ile Ala Gly Gly Thr Asn Phe Ile 1395 1400 1405 Ser Arg Leu Val Ala Ala Leu Ile Glu Val Asn Trp Ala Phe Asp Asn 1410 1415 1420 Glu Glu Val Arg Gly Leu Lys Lys Phe Phe Leu Leu Ser Ser Arg Val 1425 1430 1435 1440 Lys Glu Leu Ile Ile Lys His Lys Val Arg Asn Glu Val Met Val Arg 1445 1450 1455 Trp Phe Gly Asp Glu Glu Val Tyr Gly Met Pro Lys Leu Val Gly Leu 1460 1465 1470 Val Lys Ala Ala Thr Leu Ser Lys Asn Lys His Cys Ile Leu Cys Thr 1475 1480 1485 Val Cys Glu Asp Arg Glu Trp Arg Gly Glu Thr Cys Pro Lys Cys Gly 1490 1495 1500 Arg Phe Gly Pro Pro Met Thr Cys Gly Met Thr Leu Ala Asp Phe Glu 1505 1510 1515 1520 Glu Lys His Tyr Lys Arg Ile Phe Phe Arg Glu Asp Gln Ser Glu Gly 1525 1530 1535 Pro Val Arg Glu Glu Tyr Ala Gly Tyr Leu Gln Tyr Arg Ala Arg Gly 1540 1545 1550 Gln Leu Phe Leu Arg Asn Leu Pro Val Leu Ala Thr Lys Val Lys Met 1555 1560 1565 Leu Leu Val Gly Asn Leu Gly Thr Glu Val Gly Asp Leu Glu His Leu 1570 1575 1580 Gly Trp Val Leu Arg Gly Pro Ala Val Cys Lys Lys Val Thr Glu His 1585 1590 1595 1600 Glu Lys Cys Thr Thr Ser Met Met Asp Lys Leu Thr Ala Phe Phe Gly 1605 1610 1615 Val Met Pro Arg Gly Thr Thr Pro Arg Ala Pro Val Arg Phe Pro Thr 1620 1625 1630 Ser Leu Leu Lys Ile Arg Arg Gly Leu Glu Thr Gly Trp Ala Tyr Thr 1635 1640 1645 His Gln Gly Gly Ile Ser Ser Val Asp His Val Thr Cys Gly Lys Asp 1650 1655 1660 Leu Leu Val Cys Asp Thr Met Gly Arg Thr Arg Val Val Cys Gln Ser 1665 1670 1675 1680 Asn Asn Lys Met Thr Asp Glu Ser Glu Tyr Gly Val Lys Thr Asp Ser 1685 1690 1695 Gly Cys Pro Glu Gly Ala Arg Cys Tyr Val Phe Asn Pro Glu Ala Val 1700 1705 1710 Asn Ile Ser Gly Thr Lys Gly Ala Met Val His Leu Gln Lys Thr Gly 1715 1720 1725 Gly Glu Phe Thr Cys Val Thr Ala Ser Gly Thr Pro Ala Phe Phe Asp 1730 1735 1740 Leu Lys Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile Phe Glu Ala Ser 1745 1750 1755 1760 Ser Gly Arg Val Val Gly Arg Val Lys Val Gly Lys Asn Glu Asp Ser 1765 1770 1775 Lys Pro Thr Lys Leu Met Ser Gly Ile Gln Thr Val Ser Lys Ser Thr 1780 1785 1790 Thr Asp Leu Thr Glu Met Val Lys Lys Ile Thr Thr Met Ser Arg Gly 1795 1800 1805 Glu Phe Arg Gln Ile Thr Leu Ala Thr Gly Ala Gly Lys Thr Thr Glu 1810 1815 1820 Leu Pro Arg Ser Val Ile Glu Glu Ile Gly Arg His Lys Arg Val Leu 1825 1830 1835 1840 Val Leu Ile Pro Leu Arg Ala Ala Ala Glu Ser Val Tyr Gln Tyr Met 1845 1850 1855 Arg Gln Lys His Pro Ser Ile Ala Phe Asn Leu Arg Ile Gly Glu Met 1860 1865 1870 Lys Glu Gly Asp Met Ala Thr Gly Ile Thr Tyr Ala Ser Tyr Gly Tyr 1875 1880 1885 Phe Cys Gln Met Pro Gln Pro Lys Leu Arg Ala Ala Met Val Glu Tyr 1890 1895 1900 Ser Phe Ile Phe Leu Asp Glu Tyr His Cys Ala Thr Pro Glu Gln Leu 1905 1910 1915 1920 Ala Ile Met Gly Lys Ile His Arg Phe Ser Glu Asn Leu Arg Val Val 1925 1930 1935 Ala Met Thr Ala Thr Pro Val Gly Thr Val Thr Thr Thr Gly Gln Lys 1940 1945 1950 His Pro Ile Glu Glu Phe Ile Ala Pro Asp Val Met Lys Gly Lys Asp 1955 1960 1965 Leu Gly Ser Glu Tyr Leu Asp Ile Ala Gly Leu Lys Ile Pro Val Glu 1970 1975 1980 Glu Met Lys Ser Asn Met Leu Val Phe Val Pro Thr Arg Asn Met Ala 1985 1990 1995 2000 Val Glu Thr Ala Lys Lys Leu Lys Ala Lys Gly Tyr Asn Ser Gly Tyr 2005 2010 2015 Tyr Tyr Ser Gly Glu Asp Pro Ser Asn Leu Arg Val Val Thr Ser Gln 2020 2025 2030 Ser Pro Tyr Val Val Val Ala Thr Asn Ala Ile Glu Ser Gly Val Thr 2035 2040 2045 Leu Pro Asp Leu Asp Val Val Val Asp Thr Gly Leu Lys Cys Glu Lys 2050 2055 2060 Arg Ile Arg Leu Ser Pro Lys Met Pro Phe Ile Val Thr Gly Leu Lys 2065 2070 2075 2080 Arg Met Ala Val Thr Ile Gly Glu Gln Ala Gln Arg Arg Gly Arg Val 2085 2090 2095 Gly Arg Val Lys Pro Gly Arg Tyr Tyr Arg Ser Gln Glu Thr Pro Val 2100 2105 2110 Gly Ser Lys Asp Tyr His Tyr Asp Leu Leu Gln Ala Gln Arg Tyr Gly 2115 2120 2125 Ile Glu Asp Gly Ile Asn Ile Thr Lys Ser Phe Arg Glu Met Asn Tyr 2130 2135 2140 Asp Trp Ser Leu Tyr Glu Glu Asp Ser Leu Met Ile Thr Gln Leu Glu 2145 2150 2155 2160 Ile Leu Asn Asn Leu Leu Ile Ser Asp Glu Leu Pro Met Ala Val Lys 2165 2170 2175 Asn Ile Met Ala Arg Thr Asp His Pro Glu Pro Ile Gln Leu Ala Tyr 2180 2185 2190 Asn Ser Tyr Glu Thr Gln Val Pro Val Leu Phe Pro Lys Ile Lys Asn 2195 2200 2205 Gly Glu Val Thr Asp Ser Tyr Asp Asn Tyr Thr Phe Leu Asn Ala Arg 2210 2215 2220 Lys Leu Gly Asp Asp Val Pro Pro Tyr Val Tyr Ala Thr Glu Asp Glu 2225 2230 2235 2240 Asp Leu Ala Val Glu Leu Leu Gly Leu Asp Trp Pro Asp Pro Gly Asn 2245 2250 2255 Gln Gly Thr Val Glu Thr Gly Arg Ala Leu Lys Gln Val Val Gly Leu 2260 2265 2270 Ser Thr Ala Glu Asn Ala Leu Leu Val Ala Leu Phe Gly Tyr Val Gly 2275 2280 2285 Tyr Gln Ala Leu Ser Lys Arg His Ile Pro Val Val Thr Asp Ile Tyr 2290 2295 2300 Ser Ile Glu Asp His Arg Leu Glu Asp Thr Thr His Leu Gln Tyr Ala 2305 2310 2315 2320 Pro Asn Ala Ile Lys Thr Glu Gly Lys Glu Thr Glu Leu Lys Glu Leu 2325 2330 2335 Ala Gln Gly Asp Val Gln Arg Cys Val Glu Ala Met Thr Asn Tyr Ala 2340 2345 2350 Arg Glu Gly Ile Gln Phe Met Lys Ser Gln Ala Leu Lys Val Lys Glu 2355 2360 2365 Thr Pro Thr Tyr Lys Glu Thr Met Asp Thr Val Thr Asp Tyr Val Lys 2370 2375 2380 Lys Phe Met Glu Ala Leu Ala Asp Ser Lys Glu Asp Ile Ile Lys Tyr 2385 2390 2395 2400 Gly Leu Trp Gly Thr His Thr Ala Leu Tyr Lys Ser Ile Ser Ala Arg 2405 2410 2415 Leu Gly Gly Glu Thr Ala Phe Ala Thr Leu Val Val Lys Trp Leu Ala 2420 2425 2430 Phe Gly Gly Glu Ser Ile Ala Asp His Val Lys Gln Ala Ala Thr Asp 2435 2440 2445 Leu Val Val Tyr Tyr Ile Ile Asn Arg Pro Gln Phe Pro Gly Asp Thr 2450 2455 2460 Glu Thr Gln Gln Asp Gly Arg Lys Phe Val Ala Ser Leu Leu Ala Ser 2465 2470 2475 2480 Ala Leu Ala Thr Tyr Thr Tyr Lys Ser Trp Asn Tyr Asn Asn Leu Ser 2485 2490 2495 Lys Ile Val Glu Pro Ala Leu Ala Thr Leu Pro Tyr Ala Ala Thr Ala 2500 2505 2510 Leu Lys Leu Phe Ala Pro Thr Arg Leu Glu Ser Val Val Ile Leu Ser 2515 2520 2525 Thr Ala Ile Tyr Lys Thr Tyr Leu Ser Ile Arg Arg Gly Lys Ser Asp 2530 2535 2540 Gly Leu Leu Gly Thr Gly Val Ser Ala Ala Met Glu Ile Met Ser Gln 2545 2550 2555 2560 Asn Pro Val Ser Val Gly Ile Ala Val Met Leu Gly Val Gly Ala Val 2565 2570 2575 Ala Ala His Asn Ala Ile Glu Ala Ser Glu Gln Lys Arg Thr Leu Leu 2580 2585 2590 Met Lys Val Phe Val Lys Asn Phe Leu Asp Gln Ala Ala Thr Asp Glu 2595 2600 2605 Leu Val Lys Glu Ser Pro Glu Lys Ile Ile Met Ala Leu Phe Glu Ala 2610 2615 2620 Val Gln Thr Val Gly Asn Pro Leu Arg Leu Val Tyr His Leu Tyr Gly 2625 2630 2635 2640 Val Phe Tyr Lys Gly Trp Glu Ala Lys Glu Leu Ala Gln Arg Thr Ala 2645 2650 2655 Gly Arg Asn Leu Phe Thr Leu Ile Met Phe Glu Ala Val Glu Leu Leu 2660 2665 2670 Gly Val Asp Ser Glu Gly Lys Val Arg Gln Leu Ser Ser Asn Tyr Ile 2675 2680 2685 Leu Glu Leu Leu Tyr Lys Phe Arg Asp Ser Ile Lys Ser Ser Val Arg 2690 2695 2700 Glu Met Ala Ile Ser Trp Ala Pro Ala Pro Phe Ser Cys Asp Trp Thr 2705 2710 2715 2720 Pro Thr Asp Asp Arg Ile Gly Leu Pro Gln Asp Asn Phe His Gln Val 2725 2730 2735 Glu Thr Lys Cys Pro Cys Gly Tyr Lys Met Lys Ala Val Lys Asn Cys 2740 2745 2750 Ala Gly Glu Leu Arg Leu Leu Glu Glu Glu Gly Ser Phe Leu Cys Arg 2755 2760 2765 Asn Lys Phe Gly Arg Gly Ser Arg Asn Tyr Arg Val Thr Lys Tyr Tyr 2770 2775 2780 Asp Asp Asn Leu Leu Glu Ile Lys Pro Val Ile Arg Met Glu Gly His 2785 2790 2795 2800 Val Glu Leu Tyr Tyr Lys Gly Ala Thr Ile Lys Leu Asp Phe Asn Asn 2805 2810 2815 Ser Lys Thr Ile Leu Ala Thr Asp Lys Trp Glu Val Asp His Ser Thr 2820 2825 2830 Leu Val Arg Val Leu Lys Arg His Thr Gly Ala Gly Tyr His Gly Ala 2835 2840 2845 Tyr Leu Gly Glu Lys Pro Asn His Lys His Leu Ile Glu Arg Asp Cys 2850 2855 2860 Ala Thr Ile Thr Lys Asp Lys Val Cys Phe Leu Lys Met Lys Arg Gly 2865 2870 2875 2880 Cys Ala Phe Thr Tyr Asp Leu Ser Leu His Asn Leu Thr Arg Leu Ile 2885 2890 2895 Glu Leu Val His Lys Asn Asn Leu Glu Asp Lys Glu Ile Pro Ala Ala 2900 2905 2910 Thr Val Thr Thr Trp Leu Ala Tyr Thr Phe Val Asn Glu Asp Ile Gly 2915 2920 2925 Thr Ile Lys Pro Ala Phe Gly Glu Lys Val Thr Leu Glu Met Gln Glu 2930 2935 2940 Glu Ile Thr Leu Gln Pro Ala Val Val Val Asp Thr Thr Asp Val Ala 2945 2950 2955 2960 Val Thr Val Val Gly Glu Ala Pro Thr Met Thr Thr Gly Glu Thr Pro 2965 2970 2975 Thr Val Phe Thr Ser Ser Gly Ser Gly Leu Lys Ser Gln Gln Val Leu 2980 2985 2990 Lys Leu Gly Val Gly Glu Gly Gln Tyr Pro Gly Thr Asn Pro Gln Arg 2995 3000 3005 Ala Ser Leu His Glu Ala Ile Gln Gly Ala Asp Glu Arg Pro Ser Val 3010 3015 3020 Leu Ile Leu Gly Ser Asp Lys Ala Thr Ser Asn Arg Val Lys Thr Ala 3025 3030 3035 3040 Lys Asn Val Lys Val Tyr Arg Gly Arg Asp Pro Leu Glu Val Arg Asp 3045 3050 3055 Met Met Arg Arg Gly Lys Ile Leu Val Val Ala Leu Ser Arg Val Asp 3060 3065 3070 Asn Ala Leu Leu Lys Phe Val Asp Tyr Lys Gly Thr Phe Leu Thr Arg 3075 3080 3085 Glu Ala Leu Glu Ala Leu Ser Leu Gly Arg Pro Lys Lys Lys Asn Ile 3090 3095 3100 Thr Lys Ala Glu Ala Gln Trp Leu Leu Cys Pro Glu Asp Gln Met Glu 3105 3110 3115 3120 Glu Leu Pro Asp Trp Phe Ala Ala Gly Glu Pro Ile Phe Leu Glu Ala 3125 3130 3135 Asn Ile Lys His Asp Arg Tyr His Leu Val Gly Asp Ile Ala Thr Ile 3140 3145 3150 Lys Glu Lys Ala Lys Gln Leu Gly Ala Thr Asp Ser Thr Lys Ile Ser 3155 3160 3165 Lys Glu Val Gly Ala Lys Val Tyr Ser Met Lys Leu Ser Asn Trp Val 3170 3175 3180 Met Gln Glu Glu Asn Lys Gln Gly Asn Leu Thr Pro Leu Phe Glu Glu 3185 3190 3195 3200 Leu Leu Gln Gln Cys Pro Pro Gly Gly Gln Asn Lys Thr Ala His Met 3205 3210 3215 Val Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Met Pro Thr Ser Cys 3220 3225 3230 His Val Phe Met Gly Thr Val Ser Ala Arg Arg Thr Lys Thr His Pro 3235 3240 3245 Tyr Glu Ala Tyr Val Lys Leu Arg Glu Leu Val Glu Glu His Lys Met 3250 3255 3260 Lys Thr Leu Cys Pro Gly Ser Ser Leu Gly Arg His Asn Asp Trp Ile 3265 3270 3275 3280 Ile Gly Lys Ile Lys Tyr Gln Gly Asn Leu Arg Thr Lys His Met Leu 3285 3290 3295 Asn Pro Gly Lys Val Ala Glu Gln Leu Cys Arg Glu Gly His Arg His 3300 3305 3310 Asn Val Tyr Asn Lys Thr Ile Ser Ser Val Met Thr Ala Thr Gly Ile 3315 3320 3325 Arg Leu Glu Lys Leu Pro Val Val Arg Ala Gln Thr Asp Pro Thr Asn 3330 3335 3340 Phe His Gln Ala Ile Arg Asp Lys Ile Asp Lys Glu Glu Asn Leu Gln 3345 3350 3355 3360 Thr Pro Gly Leu His Lys Lys Leu Met Glu Val Phe Asn Ala Leu Lys 3365 3370 3375 Arg Pro Glu Leu Glu Ser Ser Tyr Asp Ala Val Glu Trp Glu Glu Leu 3380 3385 3390 Glu Arg Gly Ile Asn Arg Lys Gly Ala Ala Gly Phe Phe Glu Arg Lys 3395 3400 3405 Asn Ile Gly Glu Ile Leu Asp Ser Glu Lys Asn Lys Val Glu Glu Ile 3410 3415 3420 Ile Asp Asn Leu Lys Lys Gly Arg Asn Ile Lys Tyr Tyr Glu Thr Ala 3425 3430 3435 3440 Ile Pro Lys Asn Glu Lys Arg Asp Val Asn Asp Asp Trp Thr Ala Gly 3445 3450 3455 Asp Phe Val Asp Glu Lys Lys Pro Arg Val Ile Gln Tyr Pro Glu Ala 3460 3465 3470 Lys Thr Arg Leu Ala Ile Thr Lys Val Met Tyr Lys Trp Val Lys Gln 3475 3480 3485 Lys Pro Val Val Ile Pro Gly Tyr Glu Gly Lys Thr Pro Leu Phe Gln 3490 3495 3500 Ile Phe Asp Lys Val Lys Lys Glu Trp Asp Gln Phe Gln Asn Pro Val 3505 3510 3515 3520 Ala Val Ser Phe Asp Thr Lys Ala Trp Asp Thr Gln Val Thr Thr Lys 3525 3530 3535 Asp Leu Glu Leu Ile Arg Asp Ile Gln Lys Tyr Tyr Phe Lys Lys Lys 3540 3545 3550 Trp His Lys Phe Ile Asp Thr Leu Thr Thr His Met Ser Glu Val Pro 3555 3560 3565 Val Ile Ser Ala Asp Gly Glu Val Tyr Ile Arg Lys Gly Gln Arg Gly 3570 3575 3580 Ser Gly Gln Pro Asp Thr Ser Ala Gly Asn Ser Met Leu Asn Val Leu 3585 3590 3595 3600 Thr Met Val Tyr Ala Phe Cys Glu Ala Thr Gly Val Pro Tyr Lys Ser 3605 3610 3615 Phe Asp Arg Val Ala Lys Ile His Val Cys Gly Asp Asp Gly Phe Leu 3620 3625 3630 Ile Thr Glu Arg Ala Leu Gly Glu Lys Phe Ala Ser Lys Gly Val Gln 3635 3640 3645 Ile Leu Tyr Glu Ala Gly Lys Pro Gln Lys Ile Thr Glu Gly Asp Lys 3650 3655 3660 Met Lys Val Ala Tyr Gln Phe Asp Asp Ile Glu Phe Cys Ser His Thr 3665 3670 3675 3680 Pro Ile Gln Val Arg Trp Ser Asp Asn Thr Ser Ser Tyr Met Pro Gly 3685 3690 3695 Arg Asn Thr Thr Thr Ile Leu Ala Lys Met Ala Thr Arg Leu Asp Ser 3700 3705 3710 Ser Gly Glu Arg Gly Thr Ile Ala Tyr Glu Lys Ala Val Ala Phe Ser 3715 3720 3725 Phe Leu Leu Met Tyr Ser Trp Asn Pro Leu Ile Arg Arg Ile Cys Leu 3730 3735 3740 Leu Val Leu Ser Thr Glu Leu Gln Val Lys Pro Gly Lys Ser Thr Thr 3745 3750 3755 3760 Tyr Tyr Tyr Glu Gly Asp Pro Ile Ser Ala Tyr Lys Glu Val Ile Gly 3765 3770 3775 His Asn Leu Phe Asp Leu Lys Arg Thr Ser Phe Glu Lys Leu Ala Lys 3780 3785 3790 Leu Asn Leu Ser Met Ser Val Leu Gly Ala Trp Thr Arg His Thr Ser 3795 3800 3805 Lys Arg Leu Leu Gln Asp Cys Val Asn Ile Gly Val Lys Glu Gly Asn 3810 3815 3820 Trp Leu Val Asn Ala Asp Arg Leu Val Ser Ser Lys Thr Gly Asn Arg 3825 3830 3835 3840 Tyr Ile Pro Gly Glu Gly His Thr Leu Gln Gly Arg His Tyr Glu Glu 3845 3850 3855 Leu Val Leu Ala Arg Lys Gln Ile Asn Asn Phe Gln Gly Thr Asp Arg 3860 3865 3870 Tyr Asn Leu Gly Pro Ile Val Asn Met Val Leu Arg Arg Leu Arg Val 3875 3880 3885 Met Met Met Thr Leu Ile Gly Arg Gly Ala 3890 3895 375 base pairs nucleic acid single linear DNA (genomic) unknown 3 GTATACGAGG TTAGTTCATT CTCGTGTACA TGATTGGACA AATCAAAATC TCAATTTGGT 60 TCAGGGCCTC CCTCCAGCGA CGGCCGAGCT GGGCTAGCCA TGCCCACAGT AGGACTAGCA 120 AACGGAGGGA CTAGCCGTAG TGGCGAGCTC CCTGGGTGGT CTAAGTCCTG AGTACAGGAC 180 AGTCGTCAGT AGTTCGACGT GAGCAGAAGC CCACCTCGAG ATGCTATGTG GACGAGGGCA 240 TGCCCAAGAC ACACCTTAAC CTAGCGGGGG TCGTTAGGGT GAAATCACAC CATGTGATGG 300 GAGTACGACC TGATAGGGTG CTGCAGAGGC CCACTATTAG GCTAGTATAA AAATCTCTGC 360 TGTACATGGC ACATG 375 375 base pairs nucleic acid single linear DNA (genomic) unknown 4 GTATACGAGG TTAGCTCTTT CTCGTATACG ATATTGGATA CACTAAATTT CGATTTGGTC 60 TAGGGCACCC CTCCAGCGAC GGCCGAAATG GGCTAGCCAT GCCCATAGTA GGACTAGCAA 120 ACGGAGGGAC TAGCCGTAGT GGCGAGCTCC CTGGGTGGTC TAAGTCCTGA GTACAGGACA 180 GTCGTCAGTA GTTCGACGTG AGCACTAGCC CACCTCGAGA TGCTACGTGG ACGAGGGCAT 240 GCCCAAGACA CACCTTAACC CTGGCGGGGG TCGCTAGGGT GAAATCACAT TATGTGATGG 300 GGGTACGACC TGATAGGGTG CTGCAGAGGC CCACTAGCAG GCTAGTATAA AAATCTCTGC 360 TGTACATGGC ACATG 375 376 base pairs nucleic acid single linear DNA (genomic) unknown 5 GTATACGAGG TTAGTTCATT CTCGTATACA CGATTGGACA AATCAAAATT TTAATTTGGT 60 TCAGGGCCTC CCTCCAGCGA CGGCCGAGCT GGGCTAGCCA TGCCCATAGT AGGACTAGCA 120 AAACGGAGGG ACTAGCCATA GTGGCGAGCT CCCTGGGTGG TCTAAGTCCT GAGTACAGGA 180 CAGTCGTCAG TAGTTCGACG TGAGCAGAAG CCCACCTCGA GATGCTACGT GGACGAGGGC 240 ATGCCAAGAC ACACCTTAAC CCTAGCGGGG GTCGCTAGGG TGAAATCACA CCACGTGATG 300 GGAGTACGAC CTGATAGGGC GCTGCAGAGG CCCACTATTA GGCTAGTATA AAAATCTCTG 360 CTGTACATGG CACATG 376 229 base pairs nucleic acid single linear other nucleic acid /desc = “3′ NON-CODING REGION OF BRESCIA” unknown 6 TGAGTGCGGG TGACCCGCGA TCTGGACCCG TCAGTAGGAC CCTATTGTAG ATAACACTAA 60 TTTTTTATTT ATTTAGATAT TACTATTTAT TTATTTATTT ATTTATTGAA TGAGTAAGAA 120 CTGGTACAAA CTACCTCATG TTACCACACT ACACTCATTT TAACAGCACT TTAGCTGGAA 180 GGAAAATTCC TGACGTCCAC AGTTGGACTA AGGTAATTTC TAACGGCCC 229 227 base pairs nucleic acid single linear other nucleic acid /desc = “3′ NON-CODING REGION OF ALFORT” unknown 7 TGAGCATGGT TGGCCCTTGA TCGGGCCCTA TCAGTAGGAC CCTATTGTAA ATAACATTAA 60 CTTATTAATT ATTTAGATAC TATTATTTAT TTATTTATTT ATTTATTGAA TGAGCAAGTA 120 CTGGTACAAA CTACCTCATG TTACCACACT ACACTCATCT TAACAGCACT TTAGCTGGAG 180 GGAAAATCCT GACGTCCACA GTTGGACTAA GGTAATTTCC TAACGGC 227 244 base pairs nucleic acid single linear other nucleic acid /desc = “3′ NON-CODING REGION OF C-STRAIN” unknown 8 TGAGCGCGGG TAACCCGGGA TCTGAACCCG CCAGTAGGAC CCTATTGTAG ATAACACTAA 60 TTTTCTTTTT TTTCTTTTTT ATTTATTTAG ATATTATTAT TTATTTATTT ATTTATTTAT 120 TGAATGAGTA AGAACTGGTA TAAACTACCT CAAGTTACCA CACTACACTC ATTTTTAACA 180 GCACTTTAGC TGGAAGGAAA ATTCCTGACG TCCACAGTTG GACTAAGGTA ATTTCTAACG 240 GCCC 244 13 base pairs nucleic acid single linear other nucleic acid unknown 9 TTTTCTTTTT TTT 13 32 base pairs nucleic acid single linear other nucleic acid unknown 10 AGATTGGATC CTAAAGTATT AAGAGGACAG GT 32 35 base pairs nucleic acid single linear other nucleic acid unknown 11 TAGTCGGATC CTTAGAATTC TGCGAAGTAA TCTGA 35 

What is claimed is:
 1. A nucleotide sequence of the genome of a hybrid classical swine fever virus (CSFV) strain in which at least one nucleotide region selected from the group consisting of the nucleotide regions encoding the amino acid sequences 268-494, 691-750, 785-870, 690-870 and 690-1063 of SEQ ID No. 2 has been substituted in its entirety by the corresponding region of the genome of another non-CSFV pestivirus strain.
 2. The nucleotide sequence of claim 1, in which said other pestivirus strain is selected from the group consisting of bovine viral diarrhoea virus (BVDV) strains and border disease virus (BDV) strains.
 3. A polypeptide encoded by the nucleotide sequence of claim
 1. 4. A recombinant virus, the genome of which is derived from a member selected from the group consisting of full-length DNA copies of the nucleotide sequence of claim 1 and RNA transcripts thereof.
 5. A diagnostic composition comprising the nucleotide of claim
 1. 6. A diagnostic composition comprising the polypeptide of claim
 3. 7. A vaccine comprising the polypeptide of claim 3 and at least a carrier.
 8. A vaccine comprising the virus strain of claim 4 and at least a carrier.
 9. A nucleotide sequence of the genome of a classical swine fever virus (CSFV) strain in which at least one nucleotide region selected from the group consisting of the nucleotide regions encoding the amino acid sequences 268-494, 691-750, 785-870, 690-870 and 690-1063 of SEQ ID No. 2 has been deleted in its entirety.
 10. A polypeptide encoded by the nucleotide sequence of claim
 9. 11. A recombinant virus, the genome of which is derived from a member selected from the group consisting of full-length DNA copies of the nucleotide sequence of claim 9 and RNA transcripts thereof.
 12. A diagnostic composition comprising the nucleotide of claim
 9. 13. A diagnostic composition comprising the polypeptide of claim
 10. 14. A vaccine comprising the polypeptide of claim 10 and at least a carrier.
 15. A vaccine comprising the virus strain of claim 12 and at least a carrier.
 16. A method of distinguishing an animal naturally infected with a pestivirus field strain from a vaccinated animal, said vaccinated animal being vaccinated with a member selected from the group consisting of pestivirus polypeptides and pestivirus strains, said member being mutated by deletion or substitution of the entirety of at least one nucleotide region selected from the nucleotide regions encoding the amino acid sequences 268-494, 691-750, 785-870, 690-870 and 690-1063 of SEQ ID No. 2, said substitution being a substitution by the corresponding region of the genome of another pestivirus species, comprising the steps of: providing a test sample containing an antibody of an animal to be distinguished; contacting said test sample with a pestivirus antigen comprising the amino acid sequence which, as a result of the mutation, is absent in said pestivirus polypeptide or pestivirus strain used for vaccination and with an antibody directed against an epitope of said pestivirus antigen, and measuring competition between said antibody in said test sample and said antibody directed against an epitope of said pestivirus antigen.
 17. The method of claim 16, in which said pestivirus field strain is a classical swine fever virus (CSFV) strain and said other pestivirus strain is selected from the group consisting of bovine viral diarrhoea virus (BVDV) strains and border disease virus (BDV) strains.
 18. The method of claim 16, wherein said pestivirus antigen is a dimerized or multimerized polypeptide and said antibody directed against an epitope of said pestivirus antigen is simultaneously present in an immobilized form and in a labeled form.
 19. The method of claim 16, wherein said pestivirus polypeptide is mutated by mutation of the nucleotide region encoding the amino acid sequence 690-1063, said antigen comprises amino acid sequence 690-1063, and said epitope is located between amino acids 785 and
 870. 